Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation--ProteinChip (SELDI) technology

Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa. However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix. In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range.     

Kinetics of jump landing in agility dogs

A recent survey reported an increased risk of injury in dogs participating in agility, a competitive canine sport involving different jumping activities. The aim of this study was to quantify the kinetic parameters during jump landing for commonly used obstacle types. It was hypothesised that with increasing obstacle height, the vertical force and vertical and accelerative horizontal impulse will increase as a result of a lengthened aerial phase, a more acute landing angle and the need to convert potential into forwards kinetic energy. Simultaneous kinetic and kinematic data were recorded from 11 competition agility dogs jumping over obstacle combinations of different height and inter-obstacle distance. Speed and landing angle of the second of the two consecutive jumps were successfully controlled by obstacle height and distance between obstacles. Statistical analysis showed differences between obstacles for peak vertical force, vertical impulse and accelerative horizontal impulse (increasing values with more acute landing angles). Extremely high peak vertical force was observed in the forelimbs (4.5 times bodyweight) when landing from a hurdle jump at high speed. Further detailed studies into the consequences for internal limb structures are warranted in order to clarify how this might be related to injury.     

Oxytetracycline Pharmacokinetics in Rainbow Trout during and after an Orally Administered Medicated Feed Regimen

Abstract

The pharmacokinetic–pharmacodynamic predictor of antimicrobial activity for tetracyclines is reported to be the area under the concentration–time curve at steady state (AUC_ss) divided by the minimal inhibitory concentration of the targeted pathogen. Here, we estimate AUC_ss values for oxytetracycline (OTC) in serum of rainbow trout Oncorhynchus mykiss by using a destructive sampling study design. Seventy-two rainbow trout were fed OTC-medicated feed at 74.7 ± 1.5 mg/kg (mean ± SD) body weight (BW) by oral gavage for 10 consecutive days. Serum was collected from nine fish at 1, 3, 6, 8, 10, 12, 15, and 22 d after dosing began. Serum OTC concentrations were measured by high-performance liquid chromatography with a 0.01-μg/mL limit of detection. The average OTC AUC_ss was 29.2 μg × h/mL and was estimated using nonlinear mixed-effects modeling and bootstrap resampling techniques. The elimination half-life was estimated as 85.0 h, and the fraction of steady state achieved was estimated as 0.85. The calculated AUC_ss (24.8 μg × h/mL) following 10 d of oral dosing with 75 mg OTC/kg BW was less than the estimated AUC_ss. Results suggest that the pharmacokinetics of OTC exposure, including the AUC_ss, is better evaluated by using multiday dosimetry than by using a standard single-dose protocol.

Received September 29, 2011; accepted January 30, 2012     

Serological evidence for Babesia canis infection of horses and an endemic focus of B. caballi in Hungary

In order to evaluate the seroconversion of horses to Babesia caballi and B. canis in Hungary, blood samples were collected from 371 animals on 23 different locations of the country. The presence of antibodies to B. caballi was screened with a competitive ELISA. All 29 positive samples came from one region (the Hortobágy). The prevalence of infection did not show correlation with sexes, and reached 100% in the age group of 2-5 years. Babesia canis-specific antibodies were demonstrated by IFAT in 6.74% of animals kept in 7 regions. The titres were low or medium level (1:40 to 1:160), indicating that the horses had previously been exposed to this piroplasm, but their infection must have been limited. The highest seropositivity rate was observed in the age group of 3-4 years, and males (stallions and geldings) were significantly more frequently infected than females. However, neither B. caballi nor B. canis could be identified in the peripheral blood samples of infected horses by PCR. Since most of the B. caballi-positive horses remained negative in the B. canis IFAT, whereas seroconversion solely to B. canis was detected in several regions of the country, serological cross-reaction between the two species can be discounted. This is the first serological evidence of horses being naturally infected with B. canis, supporting the view that piroplasms are less host specific than previously thought.     

High prevalence of mycoplasmas in the genital tract of asymptomatic stallions in Austria

Mycoplasma equigenitalium and M. subdolum have been implicated in genital disorders and infertility of horses. The reported cytopathic effects of M. equigenitalium observed in vitro underscore its potential pathogenic role in reproductive dysfunction in mares. This study was initiated to determine the prevalence of mycoplasmas in the genital tract of stallions in relationship to age, clinical signs, geographic location and semen quality. For this purpose the mycoplasma flora of the genital tract of 116 stallions of the Noric breed was determined by isolation and colony immunoblotting and by polymerase chain reaction (PCR) assays. Of 438 swabs from the genital tract, pre-ejaculatory fluid and semen samples, 352 (80%) samples were positive by PCR and 125 (29%) were positive by culture. Mycoplasmas were isolated predominantly from the fossa glandis and urethra and less frequently from the penis shaft and from semen. M. equigenitalium (89 isolates) and M. subdolum (70 isolates) were the predominant species identified. M. equirhinis and M. felis were detected in 27 and 8 samples, respectively. Comparison of these isolations with clinical signs, semen quality, and age of the stallions revealed no significant correlation. However, geographical location of the stallion significantly correlated with mycoplasma detection. These results suggest that mycoplasmas are present as commensals in the genital tract of stallions. Thus, clinically healthy stallions may present a permanent reservoir for infection of mares via venereal transmission.     

Effects of barley variety fed to steers on carcass characteristics and color of meat

This study evaluated the effect of barley varieties in the diets of finishing steers on carcass composition, fat, and lean color and the fatty acid profile of subcutaneous fat. Crossbred steers (391 kg initial BW) were assigned randomly to one of five finishing diets composed primarily of corn (n = 9), Morex barley (n = 9), Steptoe barley, (n = 9), or two experimental barley varieties SM3 (n = 9) and SM5 (n = 9). Grains were cracked prior to feeding. Diets were formulated (DM basis) to be isonitrogenous (2.24% N) and isocaloric (2.01 Mcal/kg NEm and 1.35 Mcal/kg NEg). Steers were slaughtered according to industry-accepted procedures when it was visually estimated that 70% of carcasses would grade USDA Choice. After a 24-h chill at 4 degrees C, carcass quality and yield grade data were collected by trained, experienced university personnel. Objective color (L*, a*, and b*) of both the LM and subcutaneous fat were measured, and samples of subcutaneous fat were removed from the 10th- to 12th-rib region for fatty acid analysis. Diet did not affect hot carcass weight (P = 0.15), fat thickness (P = 0.58), LM area (P = 0.57), percentage of internal fat (P = 0.52), yield grade (P = 0.96), marbling (P = 0.73), or quality grade (P = 0.10). However, the LM from steers fed diets formulated with Morex and SM5 barley varieties tended to be lighter (higher L* values, P = 0.08) than the LM from steers fed the corn-based diet. Additionally, fat from steers fed corn tended to be more yellow (higher Hunter b* values, P = 0.09) than fat from steers fed barley-based diets. Although grain source had only minimal effects on the fatty acid composition of subcutaneous fat samples, pentadecanoic acid (15:0) was greater (P < 0.05) in fat from steers fed SM3 and Steptoe barley varieties than in fat from steers fed corn. Stearic acid (18:0) concentrations were higher (P < 0.05) in fat samples from steers fed corn than in those fed the experimental barley lines (SM3 and SM5). Conversely, fat samples from steers fed Steptoe and SM5 barley had greater (P < 0.05) gadoleic acid (20:1) concentrations than fat from steers fed corn or Morex variety. Although the variety/line of barley included in the finishing diet may affect LM and fat color, grain-source (barley vs. corn) had little effect on beef carcass quality and yield grades and did not greatly alter the fatty acid composition of subcutaneous fat.     

The environmental safety of eprinomectin to earthworms

A study was conducted to assess the environmental safety of the endectocide eprinomectin to the earthworm Lumbricus terrestris under conditions mimicking typical product use on pasture. The LC50 value of eprinomectin in artificial soil after 28 days of exposure is higher than the levels expected in feces from dosed cattle or in soil fertilized with manure from dosed cattle, which indicates a wide margin of safety for this compound to earthworms. However, the no-observed-effect concentration has not been established. Therefore, the current study was conducted to determine whether there would be any effects on earthworms from feces from cattle treated with the commercial formulation of eprinomectin. Feces were collected rectally from grazing cattle on Day 0 before treatment and on Days 2, 4, 7 and 14 after treatment with EPRINEX (eprinomectin) Pour-On for Beef and Dairy Cattle (Merial Limited) at 0.5 mg eprinomectin per kg bodyweight. Assays of eprinomectin B1a (the major component of eprinomectin) were 0, 0.427, 0.152, 0.0512 and 0.00185 mg kg-1 wet weight of feces (equivalent to 0, 3.34, 1.19, 0.40 and 0.010 mg kg-1 on a dry weight basis, respectively). No significant differences (p>0.05) were observed at any day post-treatment in the survival or behavioral effects of any worms fed post-dose feces relative to the worms fed control feces. All post-dose comparisons of weight changes of living earthworms to the control group were not significantly different (p>0.05), indicating that treatment of cattle with EPRINEX (eprinomectin) Pour-On for Beef and Dairy Cattle did not affect feeding or weight gain of the earthworms. The LC50 value and the results of this study establish the wide margin of safety afforded to earthworms by eprinomectin under typical usage conditions.     

Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population

Mycoplasma agalactiae causes chronic infections in small ruminants and remains endemic in many regions of the world, despite intensive and costly eradication programs. In this study, the innate genomic plasticity of M. agalactiae was exploited to design and assess a combination of molecular epidemiological tools to trace the pathogen in different geographic locations and to understand its emergence or re-emergence after eradication campaigns. For this purpose, two collections of M. agalactiae isolates, representing European outbreaks or localized endemic disease in a single region of France, were subjected to RFLP (Restriction Fragment Length Polymorphism) analyses using two sets of DNA probes (distributed across the genome and specific for the vpma gene locus), and a previously described VNTR (Variable Number Tandem Repeats) analysis. A combination of four genome-specific DNA probes and two VNTRs gave the highest discriminative power. Molecular typing revealed that, while isolates from diverse geographical origins fell into clearly different groups, the endemic disease repeatedly observed in the Western Pyrenees region over the past 30 years has been caused by a unique subtype of M. agalactiae. This indicates that the re-emergence of the pathogen after seemingly successful eradication programs is not due to the importation of exotic strains, but to the persistence of local reservoirs of infection.     

Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes

Small ruminant lentiviruses (SRLV = maedi-visna in sheep and caprine arthritis encephalitis in goats) are distributed throughout most countries of the world, particularly Europe. Laboratories from 16 European countries established collaborations within the framework of a COST (CO-operation in the field of Scientific and Technical Research) action sponsored by the European Union in order to (i) better organize their research programmes on SRLVs and (ii) to coordinate efforts to combat these two diseases. After five years, a consensus conference--the first one in the veterinary medicine field--concluded the work of this network of laboratories by reviewing the present position and discussing three important questions in the field of SRLVs: routes of transmission, consequences of infection and potential role of eradication programmes at either a European or local level, according to the situation in each country or region. This paper brings together existing information regarding these questions and identifies areas for future research.     

Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine]

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.