Western blot analyses and LD50 determinations of Toxoplasma gondii isolates

Western blot analysis of tachyzoite detergent extracts revealed striking similarities and only minor differences in the antigenic profiles of four isolates, including one new isolate of Toxoplasma gondii. At least nine common protein bands with molecular weights ranging from 14,000-108,000 were recognized by all antitoxoplasma antisera. Differences existed primarily between molecular weights 28,000 and 33,000. The LD50 determinations showed that strains of laboratory mice were affected differently by the various Toxoplasma isolates used in this study. Results indicate a need for further investigations into whether there are true strain differences of Toxoplasma gondii or simply different isolates of the same strain of this single species in the genus Toxoplasma.     

Behavior of palatine tissue during abnormal palatogenesis in the mouse (Mus musculus)

The development of the palatine process in die presence of phenylbutazone and indomethacin was carried out in preparations collected at 13.5 days of gestation, followed by tissue culture in the mouse. It was demonstrated that both agents interfere with the normal development of the palate. The mechanisms which are apparently responsible for this abnormality are discussed.     

Limitations of in situ hybridization for the detection of bluetongue virus in blood mononuclear cells

In situ nucleic acid hybridization was tested for the ability to detect bluetongue virus (BTV) nucleic acids in blood mononuclear cells. A standard protocol was devised and applied to the demonstration of BTV genetic sequences in cultured bovine mononuclear cells that had been infected in vitro. In situ hybridization using biotinylated single-stranded RNA probes, in the presence of 50% formamide at 50 C, demonstrated an intense, positive signal in 0.001-0.01% of the BTV-infected cultured mononuclear cells. The protocol was applied to isolated mononuclear cells from an experimentally infected heifer. No infected cells were observed by this method, although the blood specimens were obtained during peak viremia.     

Adhesion of outer membrane proteins containing tandem repeats of Anaplasma and Ehrlichia species (Rickettsiales: Anaplasmataceae) to tick cells

Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale, the type species for the genus Anaplasma, was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium "mucin", which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium "mucin" were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium "mucin" adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium "mucin" as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.     

Argentine strain of equine herpesvirus 1 isolated from an aborted foetus shows low virulence in mouse respiratory and abortion models

The equine herpesvirus 1 (EHV-1) was isolated in Argentina from an aborted equine foetus in 1979. This virus (SPv) has special restriction patterns (RP) in comparison with other Argentine isolates. In addition, SPv could be distinguished on the basis of its pathogenicity in baby mice inoculated intracerebrally. We studied the growth properties of the SPv in cell culture and its effects in a mouse respiratory and abortion model. We observed that SPv did not modify its capacity to grow in cell culture with respect to reference HH1 strain. Nevertheless, we found significant differences between the titres of the two strains at 8-14 h post-infection (PI). In this work we demonstrated that SPv showed low virulence in female at different stages of gestation, consistently, with results found in the mouse respiratory model. We considered that this low virulence of SPv could be related to its RP because the RP of HH1 strain are similar to those of the HVS25A strain and both showed effect on pregnant mice. More specific studies about genomic alterations to the SPv are necessary for identifying, more clearly, if the intra-strain variations have relation with the low virulence in the mouse respiratory and abortion model.     

Cardiovascular effects of insufflation of the abdomen with carbon dioxide in standing horses sedated with detomidine

OBJECTIVE: To determine the cardiovascular effects of 60 minutes of abdominal insufflation with CO2 to an intra-abdominal pressure of 15 mm Hg in standing horses receiving a constant rate infusion of detomidine. ANIMALS: 5 horses. PROCEDURE: Horses were randomly allocated into treatment or control groups. A washout period of a minimum of 7 days separated the 2 experimental periods of the crossover study. Catheters were placed into the right atrium, pulmonary artery, jugular vein, and right transverse facial artery after lidocaine infiltration. All horses were sedated with detomidine (8.54 microg/kg/h, i.v.). Horses in the treatment group received abdominal insufflation with CO2 via a laparoscopic cannula to a final and constant intra-abdominal pressure of 15 mm Hg for 60 minutes. Systemic arterial pressure, right atrial pressure, heart rate, cardiac output, core body temperature, and the pH and gas tensions of arterial and mixed venous blood were obtained. Cardiac index and systemic vascular resistance were calculated. Data were collected in 3 stages: preinsufflation (-10 and -5 minutes), insufflation (0, 15, 30, 45, and 60 minutes), and postinsufflation (70 and 80 minutes). The quality of sedation and level of analgesia were determined. RESULTS: The PaO2 of horses in the treatment group was significantly higher after 60 minutes of pneumoperitoneum than in the control group. Core body temperature decreased significantly from baseline in both groups. CONCLUSIONS AND CLINICAL RELEVANCE: A 60-minute period of abdominal insufflation to an intra-abdominal pressure of 15 mm Hg did not induce significant cardiovascular abnormalities in healthy horses.     

Per os efficacy of Ajuga extracts against sucking insects

We studied the efficacy of water-soluble extracts from four Ajuga spp on the post-embryonic development of two exopterygota (sucking insect) species. To allow comparison between different Ajuga species, results are expressed in terms of quantity of plant extracted per litre of test solution. Crude methanolic extracts of all Ajuga plants tested, with the exception of A genevensis, showed considerable per os efficacy against larvae of both Dysdercus cingulatus F and Acyrthosiphon pisum (Harris) even at 1 g litre(-1). In the aphid tests the order of efficacy was A bracteosa Wallich ex Benth > A chamaepitys Schreber > A reptans L > A genevensis L. On D cingulatus the order of efficacy was: A reptans > A bracteosa > A chamaepitys > A genevensis. Extracts were fractionated on SepPak using a range of methanol/water mixtures. Results are expressed in terms of the initial weight of plant extracted. The 100% methanolic fraction of A chamaepitys was highly effective on A pisum (100% mortality at 1 g litre(-1)) and less effective on D cingulatus (about 60% mortality at 5 g litre(-1)). The entire 60 methanol + 40 water fraction was effective against test insects but showed different efficacies according to test species and concentration applied. 20-Hydroxyecdysone (20E), cyasterone (Cy) and ajugalactone (Ajl) were identified in the fractions from all Ajuga species, but the remaining phytoecdysteroid profile was quite different between Ajuga species. Capitasterone (Cap) and 28-epi-sengosterone (5Cy28') were found only in A reptans, makisterone A (MaA) and 29-norcyasterone (29NCy) were only in A chamaepitys, while 22-acetylcyasterone (Cy22A), 3-epi-cyasterone (Cy') and 3-epi-22-acetylcyasterone (Cy'22A) were only in A bracteosa. The total amount of phytoecdysteroids was 2053 mgkg(-1) for A bracteosa, 1892 mgkg(-1) for A reptans and 95 mg kg(-1) for A chamaepitys.     

Induction of aggregation in porcine lymphoid cells by antibodies to CD46

CD46 is a major transmembrane glycoprotein that belongs to the regulator of complement activation (RCA) family. Recently, mAbs to human CD46 were shown to suppress IL-12 production. Here, we describe that mAbs against different porcine CD46 epitopes induced a marked adhesion of normal lymphocytes. Addition of low amounts of antibody to freshly isolated lymphocytes or thymocytes resulted in the clustering of the cells. Cross-linking of CD46 molecules seems essential since Fab fragments failed to induce aggregation. This aggregation was dependent on active cell metabolism and on the presence of divalent cations and required a functional cytoskeleton. It was not inhibited by antibodies to CD18, CD29, CD2, CD11a and CD11b. Staurosporine, an inhibitor of protein kinases, partially blocked the aggregation. This finding is indicative of a role of protein kinases in the transduction of the signal generated by CD46 engagement.     

Rapid field detection of Ralstonia solanacearum in infected tomato and potato plants using the Staphylococcus aureus slide agglutination test

Ralstonia solanacearum is the major cause of bacterial wilt, which affects over 200 species of plants, many of economic importance. Current methods for detection and identification of the pathogen rely on isolation on semi-selective media followed by a combination of serological and molecular techniques and plant inoculation. Such methods are time-consuming, and require extensive laboratory facilities and skilled personnel. A reliable and rapid screening technique, which could be applied in the field, would reduce the number of samples submitted for laboratory testing and provide a vital component in disease control measures. A programme for the control of R. solanacearum has been introduced in Portugal and a Staphylococcus aureus slide agglutination test was used directly on tomato and potato plants in the field and on bacterial cultures under laboratory conditions. The results obtained show that this technique can play a major role in control programmes by providing a sensitive tool for the rapid detection of the pathogen.