Serial cardiac biomarkers, pulmonary artery pressures and traditional parameters of fluid status in relation to prognosis in patients with chronic heart failure: Design and rationale of the BioMEMS study.

AIMS: Heart failure (HF), a global pandemic affecting millions of individuals, calls for adequate predictive guidance for improved therapy. Congestion, a key factor in HF-related hospitalizations, further underscores the need for timely interventions. Proactive monitoring of intracardiac pressures, guided by pulmonary artery (PA) pressure, offers opportunities for efficient early-stage intervention, since haemodynamic congestion precedes clinical symptoms. METHODS: The BioMEMS study, a substudy of the MONITOR-HF trial, proposes a multifaceted approach integrating blood biobank data with traditional and novel HF parameters. Two additional blood samples from 340 active participants in the MONITOR-HF trial were collected at baseline, 3-, 6-, and 12-month visits and stored for the BioMEMS biobank. The main aims are to identify the relationship between temporal biomarker patterns and PA pressures derived from the CardioMEMS-HF system, and to identify the biomarker profile(s) associated with the risk of HF events and cardiovascular death. CONCLUSION: Since the prognostic value of single baseline measurements of biomarkers like N-terminal pro-B-type natriuretic peptide is limited, with the BioMEMS study we advocate a dynamic, serial approach to better capture HF progression. We will substantiate this by relating repeated biomarker measurements to PA pressures. This design rationale presents a comprehensive review on cardiac biomarkers in HF, and aims to contribute valuable insights into personalized HF therapy and patient risk assessment, advancing our ability to address the evolving nature of HF effectively.

Contemporary guideline-directed medical therapy in de novo, chronic, and worsening heart failure patients: First data from the TITRATE-HF study.

AIMS: Despite clear guideline recommendations for initiating four drug classes in all patients with heart failure (HF) with reduced ejection fraction (HFrEF) and the availability of rapid titration schemes, information on real-world implementation lags behind. Closely following the 2021 ESC HF guidelines and 2023 focused update, the TITRATE-HF study started to prospectively investigate the use, sequencing, and titration of guideline-directed medical therapy (GDMT) in HF patients, including the identification of implementation barriers. METHODS AND RESULTS: TITRATE-HF is an ongoing long-term HF registry conducted in the Netherlands. Overall, 4288 patients from 48 hospitals were included. Among these patients, 1732 presented with de novo, 2240 with chronic, and 316 with worsening HF. The median age was 71 years (interquartile range [IQR] 63-78), 29% were female, and median ejection fraction was 35% (IQR 25-40). In total, 44% of chronic and worsening HFrEF patients were prescribed quadruple therapy. However, only 1% of HFrEF patients achieved target dose for all drug classes. In addition, quadruple therapy was more often prescribed to patients treated in a dedicated HF outpatient clinic as compared to a general cardiology outpatient clinic. In each GDMT drug class, 19% to 36% of non-use in HFrEF patients was related to side-effects, intolerances, or contraindications. In the de novo HF cohort, 49% of patients already used one or more GDMT drug classes for other indications than HF. CONCLUSION: This first analysis of the TITRATE-HF study reports relatively high use of GDMT in a contemporary HF cohort, while still showing room for improvement regarding quadruple therapy. Importantly, the use and dose of GDMT were suboptimal, with the reasons often remaining unclear. This underscores the urgency for further optimization of GDMT and implementation strategies within HF management.

Interference between Escherichia coli genotypes from the E. coli peritonitis syndrome given simultaneously to productive SPF White Leghorn hens by intratracheal inoculation.

RESEARCH HIGHLIGHTS: All four or only two E. coli genotypes were found in groups of hens given mixes of four genotypes.In contrast, only one genotype was found in individual hens.E. coli genotypes interfere with each other in hens after given as a mix.Interference is likely based on a random process.Broad protection can best be assessed by challenging with single genotypes.

Assessment of the best inoculation route for virulotyping Enterococcus cecorum strains in a chicken embryo lethality assay.

The study aim was to determine the best inoculation route for virulotyping Enterococcus cecorum in a chicken embryo lethality assay (ELA). Twenty-eight genetically different strains were used. Fourteen strains were isolated from cloaca swabs of broiler reproduction chickens (cloaca strains) and 14 strains from broilers with E. cecorum lesions (lesion strains). In all ELAs, 12-day incubated embryonated broiler eggs were inoculated with approximately 100 colony-forming units of E. cecorum/egg. Twenty embryos per inoculation route and strain were used in each of three experiments. In Experiment 1, four cloaca and four lesion strains were inoculated via various routes, i.e. albumen, amniotic cavity, allantoic cavity, chorioallantoic membrane, intravenous or air chamber. The albumen inoculation route showed low mortality with cloaca strains, high mortality with lesion strains and the largest difference in mortality between these groups of strains (≥60%). This route was therefore used in subsequent experiments. In Experiment 2, the same strains were used to test reproducibility, which proved to be generally good. All 28 strains were thereafter used in Experiment 3. In the three experiments, mortality caused by cloaca and lesion strains ranged from 0-25% and from 15-100%, respectively. Recovery rates, assessed in all experiments after albumen inoculation, were significantly lower from eggs inoculated with cloaca strains, compared to lesion strain-inoculated eggs (P < 0.05). However, the bacterial load of eggs with positive recovery was similar in both groups. In conclusion, the albumen inoculation route appeared to be the best to virulotype E. cecorum strains.RESEARCH HIGHLIGHTS The albumen route is the best to differentiate between E. cecorum strains.Egg albumen likely affects cloaca E. cecorum strains more than lesion strains.Based on SNPs, E. cecorum cloaca strains are clustered as well as lesions strains.

In vivo typing of Escherichia coli obtained from laying chickens with the E. coli peritonitis syndrome.

Fourteen genetically different chicken Escherichia coli strains were biotyped in hens to examine if any E. coli strain at high dose can induce the E. coli peritonitis syndrome (EPS). Moreover, biotyping was performed in embryos and the median lethal dose (LD50) of three strains was determined in hens. Nine strains were obtained from femur marrow and one strain from caecum of hens that had died from EPS. One strain originated from the inflamed pericardium of a broiler and three strains from the cloaca of specified pathogen-free (SPF) broiler breeders. Strains were inoculated intratracheally into separate groups of 32 productive SPF White Leghorn (WL) hens at a dose of 7.8-9.2 log10 colony forming units (CFU) per hen and into the allantoic cavity of separate groups of 20 SPF WL embryos incubated during 14 days in a dose of 4.2-4.6 log10 CFU per embryo. The embryo test was replicated. Bone marrow and pericardium strains induced EPS, the other strains did not. Based on mortality in hens, EPS-inducing strains could be classified as very virulent (59-100% mortality), moderately virulent (38% mortality) and low virulent (6% mortality). In productive SPF WL hens, the LD50 of three very virulent strains ranged from <2.7 to 5.3 log10 CFU. Virulent and avirulent strains killed 60-95% and 0-30% of embryos, respectively. The embryo lethality test, which showed good reproducibility, did not discriminate within virulent strains, but can nevertheless be considered as a useful alternative for biotyping E. coli in productive hens.RESEARCH HIGHLIGHTSEven at high doses, no E. coli strain could induce EPS.Substantial differences in virulence exist within very virulent E. coli strains.The embryo lethality test is a useful alternative for biotyping E. coli in laying hens.Broiler colibacillosis may represent a source of EPS strains for layers and vice versa.

Prevalence of trichomonads in the cloaca of wild wetland birds in the Netherlands.

Severe granulomatosis in productive layer chickens due to Tetratrichomonas gallinarum strain 13/16632 infection occurred in 2013 and 2017 on farms situated in a wetland area in the Netherlands. We hypothesized that wetland birds could be the source of the infection. Therefore, a prevalence study on trichomonads was performed by analysing cloaca swabs of 526 birds belonging to 13 species of wetland birds. The number of birds sampled ranged from 1 to 275 per species. Birds were sampled at 15 locations in the Netherlands. DNA extracted from the cloaca swabs was subjected to nested PCR using trichomonad-specific primers targeting the internal transcribed spacer 1 (ITS1)-5.8S rRNA-ITS2 region followed by cloning and sequencing. In nine bird species, trichomonads were detected; the overall prevalence was 9% (47/526), while the prevalence in the five species for which a substantial number of birds were examined (at least 39 per species) ranged from 4% to 24%. Three trichomonad species were found: T. gallinarum, Trichomonas tenax and Simplicimonas sp. of which T. gallinarum dominated. The virulent T. gallinarum strain 13/16632 was not detected, but closely related strains were. Phylogenetic analysis revealed that all T. gallinarum isolates belonged to two clusters within lineage 15 of Tetratrichomonas lineages. All T. tenax isolates were identical and clustered with reference strain H95, while Simplicimonas sp. isolates showed large genetic diversity. Some isolates may represent a new species of the genus Simplicimonas. We conclude that trichomonads are widespread amongst wetland birds, raising the question, amongst others, of their relevance for commercial poultry.RESEARCH HIGHLIGHTSTrichomonads occur among wild wetland birds in the Netherlands.T. gallinarum is the dominant trichomonad species in the cloaca of wetland birds.Some T. gallinarum isolates are closely related to a strain causing granulomas in layer chickens.Some isolates may represent a new species of the genus Simplicimonas.

Colchicine in Patients With Chronic Coronary Disease in Relation to Prior Acute Coronary Syndrome.

BACKGROUND: Colchicine reduces risk of cardiovascular events in patients post-myocardial infarction and in patients with chronic coronary disease. It remains unclear whether this effect is related to the time of onset of treatment following an acute coronary syndrome (ACS). OBJECTIVES: This study investigates risk for major adverse cardiovascular events in relation to history and timing of prior ACS, to determine whether the benefits of colchicine are consistent independent of prior ACS status. METHODS: The LoDoCo2 (Low-Dose Colchicine 2) trial randomly allocated patients with chronic coronary disease to colchicine 0.5 mg once daily or placebo. The rate of the composite of cardiovascular death, spontaneous myocardial infarction, ischemic stroke, or ischemia-driven coronary revascularization was compared between patients with no prior, recent (6-24 months), remote (2-7 years), or very remote (>7 years) ACS; interaction between ACS status and colchicine treatment effect was assessed. RESULTS: In 5,522 randomized patients, risk of the primary endpoint was independent of prior ACS status. Colchicine consistently reduced the primary endpoint in patients with no prior ACS (incidence: 2.8 vs 3.4 events per 100 person-years; hazard ratio [HR]: 0.81; 95% confidence interval [CI]: 0.52-1.27), recent ACS (incidence: 2.4 vs 3.3 events per 100 person-years; HR: 0.75; 95% CI: 0.51-1.10), remote ACS (incidence: 1.8 vs 3.2 events per 100 person-years, HR: 0.55; 95% CI: 0.37-0.82), and very remote ACS (incidence: 3.0 vs 4.3 events per 100 person-years, HR: 0.70; 95% CI: 0.51-0.96) (P for interaction = 0.59). CONCLUSIONS: The benefits of colchicine are consistent irrespective of history and timing of prior ACS. (The LoDoCo2 Trial: Low Dose Colchicine for secondary prevention of cardiovascular disease [LoDoCo2] ACTRN12614000093684).

Success rates of inoculation of the various compartments of embryonated chicken eggs at different incubation days.

Inoculation of embryonated chicken eggs has been widely used during the past decades; however, inoculation success rates have not been investigated systematically. In this study named success rates were assessed in brown eggs incubated between 5 and 19 days, which were inoculated with 0.2 ml methylene blue per egg. Inoculations were performed in a simple and fully standardized way. Five embryonic compartments were targeted blindly (amniotic cavity, embryo, allantoic cavity, albumen and yolk) with needles of four different lengths; albumen and yolk were targeted with eggs in upside down position. Three compartments were inoculated within sight (air chamber, chorioallantoic membrane and blood vessel). Twenty embryos were used per incubation day, intended deposition site and needle length. Success rates were assessed by visual inspection after breaking the eggs. The inoculations targeting albumen, yolk, amniotic cavity and embryo yielded low scores. Magnetic resonance imaging was performed to elucidate the reason(s) for these low success rates: needles used were of appropriate length, but embryo and amniotic cavity had variable positions in the eggs, while albumen and yolk rapidly changed position after turning the eggs upside down. The latter led to adjustment of the inoculation method for albumen and yolk. Failures to inoculate compartments within sight were immediately visible; therefore, these eggs could be discarded. Except for the amniotic cavity, full scores (20/20) were obtained for all compartments although not always on every day of incubation. In conclusion, the present study may serve as a guide to more accurately inoculate the various chicken embryo compartments. RESEARCH HIGHLIGHTS Blind inoculation of embryonated egg compartments was successful, except for the amniotic cavity. MRI showed rapid position change of albumen and yolk after turning eggs upside down. In ovo vaccination against Marek's disease might be improved by using 38 mm needles.

Randomized, double-blind, placebo-controlled, multicentre pilot study on the effects of empagliflozin on clinical outcomes in patients with acute decompensated heart failure (EMPA-RESPONSE-AHF).

AIMS: Inhibition of sodium-glucose co-transporter 2 (SGLT2) reduces the risk of death and heart failure (HF) admissions in patients with chronic HF. However, safety and clinical efficacy of SGLT2 inhibitors in patients with acute decompensated HF are unknown. METHODS AND RESULTS: In this randomized, placebo-controlled, double-blind, parallel group, multicentre pilot study, we randomized 80 acute HF patients with and without diabetes to either empagliflozin 10 mg/day or placebo for 30 days. The primary outcomes were change in visual analogue scale (VAS) dyspnoea score, diuretic response (weight change per 40 mg furosemide), change in N-terminal pro brain natriuretic peptide (NT-proBNP), and length of stay. Secondary outcomes included safety and clinical endpoints. Mean age was 76 years, 33% were female, 47% had de novo HF and median NT-proBNP was 5236 pg/mL. No difference was observed in VAS dyspnoea score, diuretic response, length of stay, or change in NT-proBNP between empagliflozin and placebo. Empagliflozin reduced a combined endpoint of in-hospital worsening HF, rehospitalization for HF or death at 60 days compared with placebo [4 (10%) vs. 13 (33%); P = 0.014]. Urinary output up until day 4 was significantly greater with empagliflozin vs. placebo [difference 3449 (95% confidence interval 578-6321) mL; P < 0.01]. Empagliflozin was safe, well tolerated, and had no adverse effects on blood pressure or renal function. CONCLUSIONS: In patients with acute HF, treatment with empagliflozin had no effect on change in VAS dyspnoea, diuretic response, NT-proBNP, and length of hospital stay, but was safe, increased urinary output and reduced a combined endpoint of worsening HF, rehospitalization for HF or death at 60 days.

Tetratrichomonas gallinarum granuloma disease in a flock of free range layers.

Granuloma disease in a flock of free range productive layers in the Netherlands in 2017 is described. The disease resembled granuloma outbreaks in layers caused by Tetratrichomonas gallinarum in 2013 and occurred in the same area in which the rearing farm considered as the source of the 2013 outbreaks was located. Between 55 and 84 weeks of age mortality was 20.3% (breeder's norm 3.9%). All dead hens examined (n = 20) showed granulomas especially in liver and ceca. Nine hens with or without liver and/or ceca granulomas were examined for trichomonads in mentioned organs by in situ hybridization (ISH), nested PCR, and cloning and sequencing. Ceca were also examined by culture. T. gallinarum ISH was positive in all livers and ceca with granulomas and negative in case granulomas were absent. T. gallinarum strain 13/16632, which caused the 2013 outbreaks was found in 4/8 hens with granulomas. Moreover, other trichomonads were detected: a T. gallinarum strain GPO-like and a Simplicimonas sp. strain GABC1-like. Mixed infections also occurred. Infectious causes of granuloma disease other than the afore-mentioned trichomonads could be excluded. Trichomonad DNA was not detected in environmental samples and wild ducks originating from the farm of concern, except for one duck in which the same Simplicimonas sp. as in hens was detected, leaving the source of the T. gallinarum infection in hens unknown. It is concluded that the herein described granuloma disease likely was caused by T. gallinarum strain 13/16632. However, the pathogenicity of the other trichomonads found remains to be clarified.

The efficacy of inactivated Escherichia coli autogenous vaccines against the E. coli peritonitis syndrome in layers.

Autogenous Escherichia coli vaccines to prevent the E. coli peritonitis syndrome (EPS) in laying hens are often used in the field, although their effectiveness has not been demonstrated yet. Therefore, in this study, which consisted of two experiments, their efficacy was assessed. In the first experiment, the EPS-inducing ability of three E. coli isolates originating from bone marrow of hens that died due to EPS and with different Pulsed-Field Gel Electrophoresis patterns, was examined by intravenous inoculation of the isolates in 17-week-old brown layers. Based on the results one isolate was chosen for the preparation of the vaccines and for homologous challenge and another one for heterologous challenge performed in the second experiment. In the named experiment, groups of laying hens which had been vaccinated intramuscularly at 14 and 18 weeks of age with inactivated vaccine either formulated as aqueous suspension or as water-in-oil emulsion were homologously or heterologously challenged per aerosol at 30 weeks of age. The vaccines contained ≥108.2 formaldehyde-inactivated colony-forming units (cfu) of E. coli per hen dose in 0.5 ml. The estimated E. coli challenge dose uptake ranged from 105.8 to 106.5 cfu per hen. Groups consisted of 18 hens each and were housed in separate isolators from 27 weeks of age. Control groups were included in this experiment, which was ended eight days after challenge. Vaccinations had no effect on body growth and both vaccine types induced (almost) complete protection against homologous challenge, while protection against heterologous challenge was inconclusive.

Coligranulomatosis (Hjärre and Wramby's disease) reconsidered.

Coligranulomatosis (Hjärre and Wramby's disease) is considered to be a disease of chickens, turkeys and partridges that occurs sporadically in individual, adult birds. Therefore, the condition is not of economic importance, but is of interest due to the similarity of its lesions to those of tuberculosis. In a number of cases the disease could be reproduced by inoculation via artificial routes of granuloma homogenate or Escherichia coli bacteria isolated from the lesions. Oral inoculations always failed. Occasionally, also serious outbreaks of granuloma disease have been reported in chickens, turkeys and quails. E. coli bacteria were either not isolated or isolated, but the disease could not be reproduced with the isolates, which means that the essence of Koch's postulates was not fulfilled. Also other evidence of causality was not presented. Therefore, these disease cases might have been wrongly diagnosed as coligranulomatosis. Instead they may have been caused by Tetratrichomonas gallinarum, a parasite, which has the ability to induce severe granulomatosis in chicken flocks as has been shown recently. It is concluded that whenever severe granuloma disease is observed in poultry flocks at a large scale and is thus economically relevant, T. gallinarum should be included and rank high in the list of differential diagnoses.

Primary Newcastle disease vaccination of broilers: comparison of the antibody seroresponse and adverse vaccinal reaction after eye-nose drop or coarse spray application, and implication of the results for a previously developed coarse dry powder vaccine.

To compare antibody seroresponse and adverse vaccinal reaction induced by Newcastle disease (ND) vaccination after eye-nose drop or coarse spray, groups of SPF broiler hens were vaccinated at day 4 (day of hatch is day 0) and intratracheally inoculated with Escherichia coli at day 11. Body weight gain (BWG) was assessed between day 4 and day 18; colibacillosis lesions and serum antibodies were determined at day 18. Meaningful comparison requires similar vaccine uptake. Vaccine virus loss during spray relative to eye-nose drop, which was assessed by comparing the results of endpoint titrations, was 3 log10. Colibacillosis lesions in birds spray vaccinated with 106.4 EID50/chicken were significantly more severe (P < 0.05), compared to those in birds eye-nose drop vaccinated with 103.4 EID50/chicken, while the seroresponse was slightly but significantly (P < 0.05) stronger. Colibacillosis lesion scores inversely paralleled BWG. It is concluded that: (1) There is room to improve the coarse ND vaccine spray used regarding adverse vaccinal reaction, while maintaining a sufficient immune response. This is also applicable to the coarse ND powder vaccine studied in previous research, which induced similar antibody response and adverse vaccinal reaction as the spray vaccine used here. (2) The vaccine virus dose influences the colibacillosis susceptibility at seven days post vaccination, as the dynamics of the vaccine virus infection is likely dose-dependent. (3) Low vaccine virus doses likely result in heterogeneous vaccine-take followed by vaccine virus spread from vaccine shedding birds to their non-vaccine virus infected flock mates ("rolling vaccinal reaction").

The incidence and economic impact of the Escherichia coli peritonitis syndrome in Dutch poultry farming.

The incidence and economic impact of the Escherichia coli peritonitis syndrome (EPS), characterized by acute mortality, were estimated in chicken egg-producing farms in the Netherlands in 2013. The incidence was significantly higher (P < 0.05) in the meat-sector (35% affected farms) compared to the layer-sector (7% affected farms). In consumption egg-producing farms EPS occurred on 12% of the free range and organic farms, while it was found on 1% and 4% of the cage and barn farms, respectively. Data from four layer and two broiler breeder flocks with EPS were used to estimate the overall economic impact of the disease. Mean numbers of eggs lost were 10 and 11 per hen housed (phh), while mean slaughter weight loss was 0.2 and 0.5 kg phh in the four layer and two broiler breeder flocks, respectively. Total losses including costs of destruction of dead hens, compensated for reduced feed intake due to a smaller flock size, ranged from €0.28 phh (cage farms) to €9.75 phh (grandparent farms) in the layer-sector and from €1.87 phh (parent farms) to €10.73 phh (grandparent farms) in the meat-sector. Antibiotics against EPS were given often and repeatedly especially in the meat-sector. Including the costs of antibiotics, total losses were estimated at €0.4 million, €3.3 million and €3.7 million for the layer-sector, the meat-sector and poultry farming as a whole, respectively. Research focusing on the prevention and treatment of EPS is justified by its severe clinical and economic impact.

Comparison of Newcastle disease vaccine administered as powder or liquid in relation to the serum antibody response and adverse vaccinal reactions in broilers.

Liquid spray and aerosol mass vaccination of poultry have several drawbacks, such as uncontrolled deposition of vaccine particles in the respiratory tract and vaccine virus inactivation by formation and evaporation of droplets. These may be addressed by using dry powder vaccines with defined particle size distribution targeting the upper (primary vaccination) or the entire respiratory tract (booster vaccination). Therefore, a coarse Newcastle disease (LZ58 strain) powder vaccine was administered to specified pathogen free (SPF) broiler hens to compare the antibody response and adverse vaccinal reactions with those induced by a coarse liquid spray and a fine liquid aerosol. Groups of 40 broilers each housed in isolators were vaccinated at 4 days of age and intratracheally inoculated with Escherichia coli (strain 506) at 11 days of age. Adverse vaccinal reactions were evaluated by measuring body weight gain and mortality between 4 and 11 days of age and between 11 and 18 days of age, and by recording colibacillosis lesions at 18 days of age. The antibody serum response was measured at 18 days of age by the haemagglutination inhibition test. Despite the relative low initial vaccine virus loss and narrow particle size distribution of the powder vaccines in comparison with their liquid counter parts, no significant differences (P > 0.05) regarding adverse vaccinal reactions and antibody response were observed between broilers vaccinated with the powder vaccines or with their liquid counterparts.

Molecular typing of avian pathogenic Escherichia coli colonies originating from outbreaks of E. coli peritonitis syndrome in chicken flocks.

Escherichia coli colonies isolated from the bone marrow of fresh dead hens of laying flocks with the E. coli peritonitis syndrome (EPS) were genotyped using pulsed-field gel electrophoresis (PFGE). Typing is important from an epidemiological point of view and also if the use of autogenous (auto)vaccines is considered. Birds with EPS originated from one house of each of three layer farms and one broiler breeder farm. Farms were considered as separate epidemiological units. In total, six flocks were examined including two successive flocks of one layer farm and the broiler breeder farm. E. coli colonies (one per bird) from nine to 16 hens of each flock were genotyped. The clonality of E. coli within birds was studied using five colonies of each of nine to 14 birds per flock. E. coli genotypes, which totalled 15, differed between farms and flocks except for two successive layer flocks that shared three genotypes. One to five genotypes were found per flock with one or two genotypes dominating each outbreak. Within hens, E. coli bacteria were always clonal. Colonies of the same PFGE type always had the same multilocus sequence type. However, four PFGE types shared sequence type 95. Neither PFGE types nor multilocus sequence types were unambiguously related to avian pathogenic E. coli from EPS. In cases where persistence of E. coli strains associated with EPS is found to occur frequently, routine genotyping to select strains for autovaccines should be considered.

The effect of the air sampling method on the recovery of Mycoplasma gallisepticum from experimentally produced aerosols.

BACKGROUND: A reliable air sampling method is a prerequisite to calculate the inhaled aerosol dose by animals exposed to the aerosol as precise as possible.[Comp]: Set abstract according to the journal style.[/Comp] OBJECTIVE: To examine if aerosol collection in a fluid medium (buffered peptone water (BPW) in the impinger) improves detection of viable mycoplasmas. Also the effect of adding Mycoplasma Experience (ME) broth and/or BPW to the aerosol fluid on aerosol titres was assessed. METHODS: Aerosols containing a Mycoplasma gallisepticum field or vaccine strain were simultaneously sampled with gelatin filters and by impinger immediately after ending aerosolization and 25 min later. RESULTS: Sampling of M. gallisepticum aerosols using the impinger did not yield higher aerosol titres compared to sampling with gelatin filters. Initial loss during generation of the field strain aerosol and the half-life time of viable mycoplasmas in the aerosol were 1.1-2.4 log10 and <4-15 min, respectively. The vaccine strain was more vulnerable compared to its field counterpart. In spite of higher aerosolized doses of the vaccine strain (10(8.0) to 10(8.1) versus 10(7.5) cfu per m(3) of air of the field strain), mycoplasmas were not recovered from the aerosols neither by gelatin filter nor by impinger. Therefore, half-life times could not be calculated. Addition of BPW to the aerosol fluid did not clearly improve the recovery of the field strain from the aerosol, while addition of ME broth and BPW did. CONCLUSION: Gelatin filters likely due to their relative high moisture content (10-14% wt/wt) are at least as useful as the impinger for the recovery of M. gallisepticum from aerosols, provided exsiccation of the filters is prevented.

Reproduction of the Escherichia coli peritonitis syndrome in laying hens.

In five experiments, each consisting of four or six groups with seven or 14 brown laying hens per group, birds were inoculated with an Escherichia coli strain, isolated from a layer with the E. coli peritonitis syndrome (EPS) by different routes between 23 and 33 weeks of age. Aerosol-exposed hens inhaled 10(5.1) to 10(6.2) colony-forming units per hen; hens inoculated by other routes received 10(7.6) to 10(9.1) colony-forming units per hen. In one experiment, one-half of the birds of each group were injected intraperitoneally with sterile egg yolk simultaneously with E. coli. Dead and surviving birds were necropsied and bacteriological examination of the bone marrow was performed. The percentage of birds with EPS that died was 89 (159/179). Nearly all dead birds showed septicaemia (155/159 = 97%), while most had septicaemia and peritonitis (126/159 = 79%). Surviving hens with EPS (20/179 = 11%) showed chronic peritonitis and inactive ovaries. Taking all experiments together, exposure of hens by the intravenous, intratracheal and intraperitoneal routes induced EPS in 84% (41/49), 80% (55/69) and 76% (16/21), respectively, while aerosol and intravaginal exposure resulted in EPS percentages of 57% (32/56) and 49% (28/57), respectively. Except for orally inoculated groups (7/56 = 13% EPS), in all other groups the EPS rates differed significantly (P <0.01) from those of the placebo-exposed groups (0/42). Neither the age of hens nor the presence of free yolk in the abdomen influenced the EPS rate. The results of the present study are suggestive of the respiratory and vaginal origin of EPS in the field.

Success rates of intrauterine inoculations of layers via the vagina.

Intrauterine inoculation of layer hens has been documented previously in the literature; however, its efficiency has only been assessed on a very small scale. Attempts were therefore made to inoculate 14 experimental groups each consisting of 10 commercial table egg-producing hens intrauterinely with methylene blue. The effect of four variables-oviposition (natural, hormonally induced or not), position of the hen at inoculation (vertical/horizontal), technique to access the uterus (three methods) and inoculation device (four types)-on the success rate of intrauterine inoculation of layers of different breed and age was studied. Immediately after inoculation, hens were euthanized and the presence of methylene blue and perforations in the oviduct were examined. Successful inoculation rates ranged from 0/10 to 10/10. The 100% success rate was obtained in birds without an egg in the uterus, which were restrained in a horizontal position and inoculated with a hard catheter after exposure of the uterovaginal junction. The second best score was obtained in hens inoculated shortly after natural oviposition, maintained in a vertical position and using a corkscrew-shaped saliva ejector after exposure of the vaginal orifice (7/10). In all other groups the success rate was 5/10 or less. Vaginal perforation occurred in none to eight birds per group. It is concluded that intrauterine inoculation of laying hens is only reliable in birds without an egg in the uterus after exposure of the uterovaginal junction. All other methods proved unreliable and often caused vaginal perforations. However, intravaginal inoculation can be performed with 100% accuracy providing the vaginal orifice is exposed.