Phenotypic and genotypic characterization of Paenibacillus larvae isolates

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease of honeybees (Apis melifera). The aim of this work was to develop a strategy for the subtyping and the epidemiological analysis of P. larvae. Phenotypic characterisation, susceptibility to several antibiotics, electrophoresis of whole bacterial proteins, rep-PCR, ribotyping and DGGE were assessed using a collection of P. larvae isolates from different Uruguayan and Argentinean locations. Results indicated that there are two P. larvae genotypes circulating in Uruguay ERIC I-BOX A (worldwide distributed) and ERIC I-BOX C (exclusively detected in Argentina until this study). These results suggest that P. larvae isolates had moved between Argentina and Uruguay, probably through the Uruguay River. Patterns of whole bacterial proteins, DGGE and ribotyping did not improve the P. larvae intraspecific discrimination. Antibiotic susceptibility assays showed that 100% isolates were OTC-sensitive and 22% (belonging to ERIC I-BOX A group) were sulfisoxazole-resistant. This work may contribute to the elucidation of basic aspects related to the epidemiology of AFB in Uruguay and in the region.     

Application of an electrometric method for measurement of blood cholinesterase activities in sheep, goats and cattle treated with organophosphate insecticides

A modified electrometric cholinesterase method has been described for use in ruminants exposed to organophosphate insecticides. The method was used to measure cholinesterase activities in the plasma and erythrocytes of sheep, goats and cattle treated with organophosphate insecticides under field conditions. The animals were treated topically to control ectoparasites with malathion (0.05%) or diazinon (0.06%). The treated animals did not suffer from adverse effects related to cholinesterase inhibition. However, 2 h after treatments, plasma and erythrocyte cholinesterase activities significantly decreased in sheep by 20% and 39% and in goats by 18% and 30%, respectively, when compared with respective untreated control values. Diazinon did not significantly affect plasma and erythrocyte cholinesterase activities in the cattle. The results suggest that the described electrometric method could be efficiently used for detecting cholinesterase inhibition in ruminants exposed to organophosphate insecticides.     

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

A hyperparasitic microsporidian infecting the salmon louse, Lepeophtheirus salmonis: an rDNA-based molecular phylogenetic study

The sea louse, Lepeophtheirus salmonis, is an obligate ectoparasitic copepod that lives on the external surface of salmonid fish. It is the most common ectoparasite of marine cage-reared salmonids, causing major economic loss to the aquaculture industry. During a sea louse monitoring programme, samples of L. salmonis were found to harbour an unreported microsporidian parasite. The microsporidian was observed in pre-adult and adult stages of both male and female copepods, with a prevalence of up to 5%. Unfixed spores were slightly pyriform in shape measuring 2.34 microm by 1.83 microm (+/- 0.01 microm) and were not observed to be enclosed by a sporophorous vesicle. The microsporidian infection was observed in all areas of the copepods' body, xenoma-like cysts forming directly under the cuticle in the epidermal tissue layer. In the present study, rDNA (530f-580r) sequence data gathered from the unidentified microsporidian parasite isolated from infected sea lice were compared with equivalents available in the databases in an attempt to identify its systematic position. The microsporidian was found to group within the phylogenetic clade containing the family Enterocytozoonidae, being most similar to members of the intranuclear genus Nucleospora. This is the first report of a hyperparasitic microsporidian infecting a caligid copepod.     

Influence of lambda-carrageenan on the release of systematic series of volatile flavor compounds from viscous food model systems

The effect of lambda-carrageenan addition level (0.1, 0.25, 0.4, and 0.5% w/w) and viscosity on the release of systematic series of aroma compounds (aldehydes, esters, ketones, and alcohols) was studied in thickened viscous solutions containing lambda-carrageenan and 10 wt % of sucrose. Air-liquid partition coefficients K (37 degrees C) of a total of 43 aroma compounds were determined in pure water and in the lambda-carrageenan solutions by static headspace gas chromatography. Mass transfer of the aroma compounds in water and in the thickened lambda-carrageenan solutions which had a wide viscosity range was assessed by dynamic headspace gas chromatography. K (37 degrees C) increased as the carbon chain increased within each homologous series. Esters exhibited the highest volatility, followed by aldehydes, ketones, and alcohols. Under equilibrium, no overall effect of lambda-carrageenan was found, except with the most hydrophobic compounds. Analysis of flavor release under nonequilibrium conditions revealed a suppressing effect of lambda-carrageenan on the release rates of aroma compounds, and the extent of decrease in release rates was dependent on the physicochemical characteristics of the aroma compounds, with the largest effect for the most volatile compounds. However, none of the effects was of a magnitude similar to the obtained changes in the macroscopic viscosity, and the suppressing effects are therefore attributable to the thickener and not the physical properties of the increasingly viscous systems.     

In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins

OBJECTIVE: To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. SAMPLE POPULATION: Mesenteric vessels from 6 clinically healthy horses. PROCEDURE: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. RESULTS: Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. CONCLUSION AND CLINICAL RELEVANCE: Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.     

Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s)

Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses.     

EHV-1 gene63 is not essential for in vivo replication in horses and mice, nor does it affect reactivation in the horse: short communication

The aim of this study was to investigate the role of immediate early gene (gene63) in the pathogenesis of equine herpesvirus 1 (EHV-1) acute and latent infections in equine and murine models. EHV-1 gene63 mutant virus (g63mut) along with EHV-1 (Ab4) was used for intracerebral and intranasal infection of 3 and 17-day-old mice. Both viruses were recovered at the same frequency from tissues after infection. Two Welsh ponies were infected via the intranasal route with each of the viruses. Acute infection was monitored by virus isolation from nasal swabs and peripheral blood leukocytes. Six weeks post infection, peripheral blood leukocytes were taken from ponies and in vitro reactivation was positive for both viruses. At autopsy, both viruses were isolated by co-cultivation from bronchial and submandibular lymph nodes. These findings indicate that the mutation of EHV-1 gene63 does not play a role in the establishment and reactivation from latency.     

Breed risk of pyometra in insured dogs in Sweden

An animal insurance database containing data on over 200,000 dogs was used to study the occurrence of pyometra with respect to breed and age during 1995 and 1996 in Swedish bitches <10 years of age. A total of 1,803 females in 1995 and 1,754 females in 1996 had claims submitted because of pyometra. Thirty breeds with at least 800 bitches insured each year were studied using univariate and multivariate methods. The crude 12-month risk of pyometra for females <10 years of age was 2.0% (95% confidence interval = 1.9-2.1%) in 1995 and 1.9% (1.8-2.0%) in 1996. The occurrence of pyometra differed with age, breed, and geographic location. The risk of developing pyometra was increased (identified using multivariate models) in rough Collies, Rottweilers, Cavalier King Charles Spaniels, Golden Retrievers, Bernese Mountain Dogs, and English Cocker Spaniels compared with baseline (all other breeds, including mixed breed dogs). Breeds with a low risk of developing the disease were Drevers, German Shepherd Dogs, Miniature Dachshunds, Dachshunds (normal size), and Swedish Hounds. Survival rates indicate that on average 23-24% of the bitches in the databases will have experienced pyometra by 10 years of age. In the studied breeds, this proportion ranged between 10 and 54%. Pyometra is a clinically relevant problem in intact bitches, and differences related to breed and age should be taken into account in studies of this disease.