Search

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.

In 2013, seven outbreaks of granuloma disease occurred in Dutch flocks of productive layers housed on different farms. These outbreaks were characterized by increased mortality and high incidence of granulomas, mainly in caeca (340/408 hens = 83%) and livers (69/408 hens = 17%). Mortality started to increase between 21 and 35 weeks of age and reached 3.7% to 11.0% exceeding the breeder's norm in periods ranging from 9 to 48 weeks. Some flocks also showed decreased egg production and/or loss of mean egg weight. All affected flocks were linked to one rearing farm, which therefore seemed to be the source of the disease. However, no signs of disease had been observed at this rearing farm. Sentinel hens placed in one of the affected flocks to determine whether the disease had an infectious nature developed granulomas identical to those seen in the outbreaks. Next, by fulfilling Koch's postulates it was shown that Tetratrichomonas gallinarum was the aetiological agent of the granuloma disease. The condition was reproduced in mature specified pathogen free White Leghorn hens (GD - Animal Health, Deventer, the Netherlands) by inoculation via both an artificial and a natural route with a well-defined axenic T. gallinarum isolate obtained from one of the affected flocks. Other causes of granuloma disease were excluded.

To gain more insight into the within flock transmission of Histomonas meleagridis, the shedding of parasites was quantified by a newly developed real-time quantitative (q)PCR and the basic reproduction number (R0) and the mean number of secondary infections per infectious bird per day in a susceptible population (β) of H. meleagridis in the absence of heterakis were assessed. Forty turkeys were divided into two groups of 10 and 30 birds at 14 days of age. Birds of the first group were inoculated with 200,000 histomonads each, the second group served as a susceptible contact group. Cloacal swabs were taken at -1, 1, 4, 7, 9, 11, 14, 18 and 21 days post inoculation (p.i.) to assess the shedding of the parasite by the qPCR (detection limit 330 histomonads/ml droppings). The experiment ended at 28 days p.i. Mortality was 100% in the inoculated birds and started at day 12 p.i., while in the contacts, it was 83% and started at 16 days p.i. Shedding started 1 day after the inoculation in both groups. The mean shedding levels (and 95% CI) expressed as parasite equivalents per gram cloacal content on a log10 scale in the inoculated, contact birds that died and contact birds alive were 2.0 (1.6-2.4), 1.6 (1.4-1.9) and 1.2 (0.5-2.0), respectively. Birds that died shed histomonas more often and were infectious for 13.4 days; in contrast, those that recovered were infectious for 5.7 days. R0 was estimated to be 8.4 and β 0.70. Simulations made with the parameters obtained were in agreement with the experimental results, confirming their validity.

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.

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.

Background: Malignant bowel obstruction (MBO) is a common condition among palliative patients and has limited management options. There is a paucity of widely accepted national and international evidence-based guidelines to direct the management of MBO in palliative patients., Objective: The aim of this study was to survey current practice in New Zealand of nonopioid pharmacological management of MBO and compare it with the available literature., Methods: The study utilized a survey consisting of three scenarios: probable incomplete malignant bowel obstruction (PIMBO), incomplete malignant bowel obstruction (IMBO), and complete malignant bowel obstruction (CMBO). An online survey was conducted over 2 months targeting palliative medicine practitioners working in a hospital, hospice inpatient unit, or community setting in New Zealand., Results: Forty-eight doctors responded. Of the respondents, 56.3% used guidelines to manage MBO; however, 88.9% of the guidelines used were locally generated at the doctor's institution. Metoclopramide was the drug of choice in treating PIMBO, whereas haloperidol and hyoscine butylbromide (HB) were the preferred drugs as the severity of bowel obstruction increased. Dexamethasone was accepted as standard practice for managing all severities of MBO. A variation in the preferred starting and maximum dose of all the drugs was seen. There was a decrease in the use of laxatives and enemas as the severity of MBO increased., Conclusion: Variation in practice for managing MBO was evident among doctors in New Zealand. There is a need for national and international evidence-based guidelines to help define best management for the differing severities of this problematic condition.

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.

Molecular typing techniques with sufficient discriminatory power are required to better understand the transmission of Mycoplasma synoviae, a poultry pathogen with increasing clinical and economic relevance. A promising molecular technique is polymerase chain reaction and subsequent sequencing based on the conserved 5' region of the M. synoviae variable lipoprotein and haemagglutinin (vlhA) gene. This technique was used for genotyping 27 mainly Dutch M. synoviae isolates from different organs of various categories of poultry housed on different farms and collected during a period of 10 years. The obtained vlhA sequences were compared with those of M. synoviae strains from Genbank and data obtained by amplified fragment length polymorphism (AFLP). Grouping based on 100% similarity revealed nine genotypes. Some isolates had identical vlhA gene sequences although they originated from different geographical areas, different years and organs. AFLP analysis results largely confirmed the results obtained by vlhA sequence typing. Our findings raise concern regarding the discriminatory power of these techniques for its use in molecular epidemiology of Dutch M. synoviae isolates and for the differentiation between M. synoviae vaccine strains and field isolates, and indicate that molecular typing based on additional markers should be considered.

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.

A quantitative polymerase chain reaction (qPCR) was validated for the detection of Mycoplasma synoviae (PCR equivalents of colony-forming units [CFU eq.]) in chicken joint specimens with time and compared with direct mycoplasma culture. Joint specimens were obtained from 70 layer pullets inoculated intravenously with M. synoviae at 6 weeks of age. Ten control birds were injected intra-articularly with Freund's complete adjuvant. Macroscopic joint lesions were observed in 54 infected birds, of which 11 showed positive M. synoviae culture. The specificity of direct mycoplasma culture was high (100%, 95% confidence interval [CI] = 74 to 100), but its sensitivity low (16%, 95% CI = 8 to 26). Most positive results were obtained during the first 2 weeks after onset of joint swelling using synovial fluid. The qPCR was positive in 26 of 28 synovial fluid samples and in 51 of 70 joint swabs. The sterile joint samples obtained from Freund's complete adjuvant-injected birds were negative in the mycoplasma culture. The specificity and sensitivity of the qPCR for synovial fluid samples were 100% (95% CI = 65 to 100) and 93% (95% CI = 77 to 99); for joint swabs they were 100% (95% CI = 74 to 100) and 73% (95% CI = 61 to 83), respectively. Positive qPCR results (10(0.3) (to) (4.6) CFU eq./ml) were found until the end of the experiment (12 weeks post inoculation). At the end of the study, eight out of 16 joint swabs from birds without macroscopic joints lesions were positive in the qPCR (10(2.0) (to) (2.8) CFU eq./ml). Under the conditions of this study, the sensitivity of the qPCR was higher than that of direct mycoplasma culture (P< 0.0001) during the acute, subacute and chronic stages of arthritis.

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.

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.

Background: Besides the anticoccidial drug resistance problem, increasing consumer concerns about food safety and residues have propelled the quest for alternative prevention and control strategies amongst which phytotherapy has gained appeal due to a renewed interest in natural medicine., Objective: The objective was in vivo screening of four phytochemicals/extracts and a fungal immunomodulatory protein (FIP) against an Eimeria acervulina infection in broilers., Animals and Methods: Four phytochemicals/extracts (extract from Echinacea purpurea, betaine (Betain™), curcumin, carvacrol (two different doses)), and a recombinant FIP from Ganoderma lucidum cloned and expressed in Escherichia coli were investigated for their anticoccidial potential. The experiment was conducted in a battery cage trial with 54 cages of eight birds each. Broilers infected with E. acervulina (a low and high infection dose of 10(4) and 10(5) sporulated oocysts, respectively) and treated with the phytochemicals/extracts or the FIP were compared with broilers treated with the anticoccidial salinomycin sodium (Sacox®) and with an untreated uninfected and an untreated infected control group. Coccidiosis lesion scores, body weight gains and oocyst shedding were used as parameters., Results: The results showed a coccidiosis infection dose effect on the mean coccidiosis lesion scores. The phytochemicals/extracts and the FIP failed to reduce coccidiosis lesion scores and oocyst shedding, while salinomycin efficiently controlled the E. acervulina infection and enabled significantly higher body weight gains., Conclusion: In conclusion, the selected phytochemicals/extracts and the FIP did not reduce the lesions of an experimentally induced E. acervulina infection.

Five phytochemicals/extracts (an extract from Echinacea purpurea, a β-glucan-rich extract from Shiitake, betaine [Betain™], curcumin from Curcuma longa [turmeric] powder, carvacrol and also a recombinant fungal immunomodulatory protein [FIP] from Ganoderma lucidum) cloned and expressed in Escherichia coli were investigated for their anticolibacillosis potential in three chicken experiments, which were conducted in floor pens. Birds that were inoculated with E. coli intratracheally were treated with the phytochemicals/extracts or the FIP and compared with doxycycline-medicated and non-medicated infected broilers. Non-medicated and non-infected birds were used as negative controls. Mortality, colibacillosis lesions and body weight gains were used as parameters. Considering the sum of dead birds and chickens with generalized colibacillosis per group, there was no significant difference between the positive control groups and birds treated with phytochemicals/extracts or the FIP. In contrast, doxycycline-treated birds showed significantly lower mortality and generalized colibacillosis. Moreover, none of the phytochemicals/extracts and the FIP improved recovery from colibacillosis lesions, while all doxycycline-treated broilers recovered completely. The negative control birds and doxycycline-treated groups consistently showed the highest weight gains. Pulsed-field gel electrophoresis of reisolates showed that they were genetically indistinguishable from the inoculation strain. In conclusion, none of the tested phytochemicals/extracts and the FIP significantly reduced the E. coli-induced mortality and generalized colibacillosis, and nor did they improve recovery from colibacillosis lesions.

Dry powders containing a live-attenuated Newcastle disease vaccine (LZ58 strain) and intended for mass vaccination of poultry were prepared by spray drying using mannitol in combination with trehalose or inositol, polyvinylpyrrolidone (PVP) and/or bovine serum albumin (BSA) as stabilizers. These powders were evaluated for vaccine stabilizing capacity during production and storage (at 6 °C and 25 °C), moisture content, hygroscopicity and dry powder dispersibility. A mixture design, varying the ratio of mannitol, inositol and BSA, was used to select the stabilizer combination which resulted in the desired powder properties (i.e. good vaccine stability during production and storage, low moisture content and hygroscopicity and good dry dispersibility). Inositol-containing powders had the same vaccine stabilizing capacity as trehalose powders, but were less hygroscopic. Incorporation of BSA enhanced the vaccine stability in the powders compared to PVP-containing formulations. However, increasing the BSA concentration increased the hygroscopicity and reduced the dry dispersibility of the powder. No valid mathematical model could be calculated for vaccine stability during production or storage, but the individual experiments indicated that a formulation combining mannitol, inositol and BSA in a ratio of 73.3:13.3:13.3 (wt/wt) resulted in the lowest vaccine titre loss during production (1.6-2.0 log(10) 50% egg infectious dose (EID(50)) and storage at 6 °C (max. 0.8 log(10) EID(50) after 6 months) in combination with a low moisture content (1.1-1.4%), low hygroscopicity (1.9-2.1% water uptake at 60% relative humidity) and good dry dispersibility properties., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: In case of persistent and sterile inflammation, anti-inflammatory drugs should be considered as first choice treatment instead of antibiotics., Objective: To assess the effect of anti-inflammatory drugs on lesions due to colibacillosis., Animals & Methods: Five groups of day-old broilers of 15 birds each were housed in isolators and were inoculated at 29 days of age with Infectious Bronchitis Virus strain M41 by the oculo-nasal and IT (intratracheal) route (10(5.4) EID(50) (egg infectious dosis 50)/broiler) and at 33 days of age with Escherichia coli strain 506 by the IT route (10(7.6) colony forming units/broiler). Broilers of four groups were treated from day 28 up to and including day 39 orally on a daily basis with either carbasalate calcium (4 × 12.5 mg), meloxicam (2 × 0.5 mg), acetaminophen (4 × 2.5 mg), or dexamethasone (1 × 1.0 mg). The fifth group was placebo-medicated. At 40 days of age, the experiment was ended and at post-mortem examination, colibacillosis lesions were assessed., Results: All broilers in the dexamethasone group died. This mortality exceeded significantly (p < 0.05) that of the other groups in which mortality ranged from 2 to 5. Mean lesion score of surviving broilers of medicated groups ranged from 5.3 to 5.8 compared to 3.9 in the placebo group and did not differ significantly between groups., Conclusion: None of the anti-inflammatory drugs had a positive effect on colibacillosis lesions., Clinical Importance: Anti-inflammatory drugs cannot be considered as an alternative for antibiotic treatment.

Four meat turkey farms with a history of lameness were investigated for the presence of Mycoplasma synoviae by testing one flock per farm for antibodies with the rapid plate agglutination (RPA) test and/or for M. synoviae DNA by polymerase chain reaction (PCR). Flocks were sampled every 2 weeks from 6 weeks of age until slaughter. If PCR results were positive, tracheal swabs were taken for mycoplasma isolation and swollen joints were sampled for general bacteriology, mycoplasma culture and virology. In one flock, all M. synoviae diagnostic tests were negative but reovirus was isolated. In the other flocks, M. synoviae was the only pathogen associated with lameness due to joint disease. M. synoviae RPA tests became positive 6 to 8 weeks later than PCR. An experimental infection was then conducted in male meat turkeys in which a negative control group was sham inoculated at 8 weeks of age, while three other groups were inoculated intravenously with M. synoviae. Turkeys in group LCh were given 10(5) colony-forming units (CFU) of an arthropathic M. synoviae chicken strain at 8 weeks; group LHCh was given a low (10(5) CFU) dose at 8 weeks, followed by a high (10(8) CFU) dose at 12 weeks, of the same chicken strain; and group HTu was inoculated with 10(8) CFU of a M. synoviae turkey joint isolate from the field study. Post-mortem examination, histopathology, serology, bacteriology and mycoplasma culture were performed at 19 weeks of age. A dose effect was found after comparing the LCh and the LHCh groups. No significant difference was observed between the HTu (10(8) CFU/bird) and the LCh (10(5) CFU/bird) group regarding the number of turkeys with arthritis, the number of M. synoviae reisolations and the mean microscopic lesion scores of joints, indicating that the M. synoviae chicken strain has greater arthropathogenic potential and that infection of turkeys in the field with such a strain may possibly have a greater clinical and economic impact.

Increased mortality and problems with lameness were reported in Dutch broiler flocks from the year 2008 onwards. Therefore, a field inventory, including 10 affected broiler flocks, nine corresponding broiler breeder flocks and five hatcheries, was carried out. The onset of clinical signs (lameness and increased mortality) started at about 2 weeks of age. The flock mortality varied from 3.1 to 8.1% at slaughter. Post-mortem lesions of broiler flocks were characterized by the occurrence of pericarditis/hydropericardium, arthritis and femoral head necrosis. Enterococcus cecorum was isolated from approximately 30% of the lesions. In the broiler breeders, E. cecorum was not isolated from any of the lesions. However, it was isolated from 31 out of 65 (47%) cloacal swabs, from two out of 65 (3%) oviduct samples, from one out 65 (1.5%) bone marrow samples and from two out of 25 (8%) blood samples. E. cecorum was not isolated from the air samples or dead-in-shell originating from the hatcheries involved. In total, 78 isolates were subjected to further typing by means of tRNA intergenic spacer PCR and confirmed as E. cecorum. The genetic relatedness of these cocci was subsequently studied using pulsed-field gel electrophoresis. The banding patterns of approximately 68% of E. cecorum isolates originating from parent stock flocks were clonal to one or more isolates of the same or other parent flocks. In contrast, isolates originating from their diseased offspring showed much greater genetic variation. Therefore, the vertical transmission of E. cecorum could not be demonstrated.

Coccidiosis in chickens is a parasitic disease with great economic significance, which has been controlled successfully for decades using mainly anticoccidial products. However, large-scale and long-term use of anticoccidial drugs has led to the worldwide development of resistance against all these drugs. In order to minimize the occurrence of resistance, the rotation of various anticoccidial drugs in single and/or shuttle programmes is used. Unfortunately, this has not solved the anticoccidial resistance problem. Recently, live anticoccidial vaccines have been incorporated into rotation programmes, resulting in an increasing incidence of anticoccidial drug-sensitive Eimeria spp. field isolates, which may ameliorate the efficacy of anticoccidial drugs. Nevertheless, possible upcoming bans restricting the use of anticoccidials as feed additives, consumer concerns on residues and increasing regulations have prompted the quest for alternative coccidiosis control strategies. Although management and biosecurity measures could halt the introduction of Eimeria spp. to a farm, in practice they do not suffice to prevent coccidiosis outbreaks. Phytotherapy, aromatherapy and pre- and probiotics either show conflicting, non-consistent or non-convincing results, and have therefore not been applied at a large scale in the field. So far, live attenuated and non-attenuated anticoccidial vaccines have proved to be the most solid and successful coccidiosis prevention and control strategy. Despite the drawbacks associated with their production and use, their popularity is increasing. If with time, the immunogenicity of subunit vaccines can be improved, they could represent the next generation of highly efficient and low-cost anticoccidial strategies.

Histomonosis, a parasitic disease of galliformes and sporadically of other birds caused by Histomonas meleagridis, can result in very high mortality, especially in turkeys. The ban on the last antihistomonal drug prompted an urgent search for alternative prevention and treatment strategies. As both paromomycin and tiamulin have been reported to have antihistomonal activity, these antibiotics were investigated in vitro by adding two-fold serial dilutions ranging from 12.5 to 400 microg/mL to cultures of H. meleagridis. Controls (no antibiotics, or 12.5 microg or 400 microg/mL dimetridazole) were included. Parasites were counted after 3, 20, 28, 44, 51, and 71 hours of incubation. Tiamulin did not have a clear antihistomonal effect, but paromomycin had an inhibitory effect at all concentrations tested. The latter antibiotic was subsequently examined in an in vivo study. Five groups of 20 1-day-old poults, matched by weight and sex, were either not treated (infected and uninfected control groups) or treated with paromomycin (100, 200, or 400 ppm) added to their feed. After 2 weeks all groups, except for the uninfected control group, were intracloacally inoculated with 200,000 histomonads per bird. A clear dose-response effect was found for paromomycin. In the 100-ppm paromomycin group, mortality was similar to that in the untreated control group, whereas about half of the birds died in the 200-ppm paromomycin group; almost complete protection against histomonosis was seen in the 400-ppm paromomycin group. This study shows that paromomycin supplied in feed at 400 ppm is a potentially preventive strategy against H. meleagridis.

Histomona meleagridis is a protozoan parasite that may cause outbreaks of histomonosis with high mortality, especially in turkey flocks. Chickens are less susceptible to the disease than are turkeys, but are considered to act as an important reservoir. To determine the seroprevalence of H. meleagridis in Dutch layer chicken flocks, a large scale seroepidemiologic study (3376 samples) was performed by sampling 12 organic flocks, 24 free-ranging flocks, 40 flocks with floor housing, and 40 flocks with cage housing. At the end of the laying period, approximately 30 blood samples per flock were collected for serology. The seroprevalence found was high. In every flock, at least one of the samples tested positive while in 87% of the flocks, at least one of the samples was strongly positive. There were no significant statistical differences in seropositivity between the housing types. To confirm the enzyme-linked immunosorbent assay (ELISA) results, a small-scale seroepidemiologic study (576 samples) was performed in 29 additional layer chicken flocks kept in different housing systems. Subsequently, a subset of five seropositive flocks was selected. Five birds were obtained from each of these flocks in order to detect the parasite using culture and PCR. In all five flocks, H. meleagridis was either isolated from (culture), detected in (PCR), or both, the birds sampled. Together with the previously performed validation studies, the latter results confirm that the positive ELISA serology found is genuine. We conclude that the seroprevalence of H. meleagridis in layers is, as anticipated, high.

Background: Enterococcus hirae-associated endocarditis, characterized by a peak in mortality during the second week of the grow-out, and occasionally lameness, was diagnosed at Dutch broiler farms., Objectives: Field cases were studied to increase knowledge on clinical and pathological characteristics, pathogenesis and epidemiology of these infections., Animals and Methods: In total, 1266 birds of 25 flocks from 12 farms were examined. Post-mortem examinations, bacteriology, histopathology, PCR and DNA fingerprinting was carried out. Six flocks were followed longitudinally (n = 1017 birds)., Results: Average mortality was 4.1% for the entire grow-out, of which 36% was attributed to endocarditis. Fibrinous thromboendocarditis of the right atrioventricular (AV) valve was found in 24% of hearts, compared to 7% and 4% with lesions of left and both AV valves, respectively. Thrombotic lesions were found in 24% (n = 432) of lungs, but only in larger branches of the Arteria pulmonalis. Occasionally, thrombi were found in the Arteria ischiadica externa and in liver and brain vessels. Enterococcus was cultured from 54% (n = 176) of heart and in 75% (n = 28), 62% (n = 106) and 31% (n = 16) of liver, bone marrow and lung samples, respectively. Further identification, using the Rapid ID Strep 32 API system and a PCR targeting mur-2 and mur-2(ed) genes was carried out on a subset of Enterococcus positive isolates (n = 65): both techniques identified the isolates as Enterococcus hirae. Pulsed-field gel electrophoresis did not indicate evidence of clonality between farms and flocks., Conclusions: The relevance of these findings for pathogenesis and epidemiology of E. hirae infections is discussed. CLINICAL IMPORTANCE. This study may facilitate diagnosis of field cases and may contribute to the design of further research and development of control measures.

The use of garlic as a treatment against helminth infections is increasing in organic layer farms in several European countries. Its efficacy against these parasites, however, has not been demonstrated thus far. Therefore, a study was conducted to determine the efficacy of a commercially available garlic product consisting of a high concentration of allicin (i.e., the main active component of garlic) against experimentally induced Ascaridia galli infection in chickens. In total, 450 Lohmann LSL-Classic cockerels were used. Group 1, the uninfected, untreated group, consisted of 50 chickens. Groups 2 to 5, each consisting of approximately 100 chickens, were inoculated with 300 embryonated A. galli eggs/chicken at 6 wk of age. Group 2 was not treated, whereas groups 3 through 5 were given daily individual oral treatments from 13 wk of age onward. Group 3 received the recommended dose of allicin for 2 wk, whereas group 4 received a 10-fold dose of allicin. Group 5 was given 10 mg of flubendazole/kg of BW for 1 wk. Necropsy of 20 birds of all groups was performed weekly between 13 and 16 wk of age to determine adult worm loads. Group 1 remained free of A. galli. The experimental infection in the other groups resulted in a mean adult worm load of approximately 16 worms/bird. No significant differences were observed in worm counts of the allicin-treated groups (groups 3 and 4) compared with the infected, untreated group (group 2) at any week (P > 0.05). In contrast, no worms were found in chickens after flubendazole treatment (group 5). It was concluded that allicin does not represent an alternative to flubendazole for the treatment of A. galli infections in chickens.

Histomonosis is an infectious disease of mainly galliform birds that can cause high mortality, especially in commercial turkey flocks. Several diagnostic tools were available to detect its causative agent, the flagellated protozoan Histomonas meleagridis. However, serological tools were largely lacking. Recently, an indirect ELISA has been described, but this test was not validated for specificity and might suffer from cross-reactivity with related protozoa. Therefore, a specific blocking-ELISA for the detection of antibodies against H. meleagridis in chicken and turkey sera was developed. For this purpose, monoclonal antibodies were raised against a detergent extracted protein of H. meleagridis. These MAbs bound to morphologically identified histomonads in liver tissue of an infected turkey. The MAbs were conjugated with horseradish peroxidase, after which the most reactive conjugate was used to set-up the blocking-ELISA. Experimentally infected turkeys (n=9) and chickens (n=10) seroconverted in the blocking-ELISA within 2-4 weeks following inoculation with a H. meleagridis field strain. The MAb did not bind to Tetratrichomonas gallinarum antigen and experimentally inoculated T. gallinarum seropositive layer chickens (n=18) showed the same inhibition percentages in the blocking-ELISA as negative control birds did. Therefore, it was concluded that H. meleagridis blocking-ELISA does not cross-react with T. gallinarum, which is a closely related protozoan frequently occurring in poultry. The repeatability and reproducibility of the H. meleagridis blocking-ELISA were high. The new blocking-ELISA is a promising tool for experimental studies; however, further validation of this test with field samples is necessary before it can be used for diagnostic purposes., (Copyright 2010 Elsevier B.V. All rights reserved.)

Recently, the causal relationship between eggshell apex abnormalities (EAA) and Mycoplasma synoviae was described. This eggshell pathology has only been documented in table egg layers both spontaneously and experimentally infected with M. synoviae, suggesting that meat-type layers are less prone to this condition. In this study the susceptibility of specified pathogen free (SPF) broiler breeder hens to produce eggs with EAA after M. synoviae infection was assessed. Five groups of 12 hens each were made: a negative control group, a group inoculated intratracheally (i.t.) with a M. synoviae EAA strain at 19 weeks of age, a group inoculated i.t. with this strain at 19 and 26 weeks of age, a group inoculated with M. synoviae i.t. at 19 weeks of age and infected 5 days earlier with infectious bronchitis virus D1466 (IBV), and a fifth group similar to the former but inoculated i.t. twice with an M. synoviae EAA strain at 19 and 26 weeks of age. Eggs with EAA were only produced after a single i.t. inoculation with the M. synoviae EAA strain if preceded by an infection with IBV. The production of eggs with EAA started 6 weeks after M. synoviae EAA inoculation and the proportion of eggs with EAA during the experiment was 9/449 (2%), which was much lower than that in SPF layer hens (14-22%). The present results suggest that broiler breeder hens are less susceptible to producing eggs with EAA after an infection with a M. synoviae EAA strain preceded by an IBV infection, compared with table egg layers. Similar to SPF egg layers, the mean daily egg production per hen was significantly reduced by the M. synoviae EAA strain and there was a general negative effect on eggshell strength by this strain, suggesting it could also have a detrimental effect on hatching egg quality.

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.

An experimental study was conducted to assess the effect of a live Mycoplasma synoviae vaccine (Vaxsafe MS; Bioproperties Pty Ltd, Ringwood, Victoria, Australia) on M. synoviae-induced eggshell apex abnormalities (EAA). Four experimental groups of specified-pathogen-free white laying hens were made. All groups were inoculated with infectious bronchitis virus D1466 at 18 weeks of age. One group did not receive further treatment (non-vaccinated non-challenged (NVNC)). Two groups were vaccinated at 14 weeks of age against M. synoviae, and one of these groups was also challenged with an EAA-inducing M. synoviae strain 5 days after infectious bronchitis virus challenge (vaccinated non-challenged (VNC) and vaccinated challenged group (VC), respectively). The fourth group was not vaccinated but was challenged with M. synoviae (non-vaccinated challenged (NVC)). Eggs with EAA eggs were produced only in the NVC and VC groups. However, the proportion of eggs with EAA and the mean daily production of eggs with EAA per chicken was significantly lower (P<0.05) in the VC group (88/741 (11.9%) and 0.09+/-0.01 eggs per hen) compared with the NVC group (148/646 (22.9%) and 0.14+/-0.01 eggs per hen). The mean daily egg production per chicken was significantly lower in the NVC group (0.48+/-0.03 eggs) compared with that of the NVNC group (0.60+/-0.03 eggs), but not significantly different from other groups. The eggshell strength of eggs with EAA (22.8 N) was significantly lower (P<0.05) than non-affected eggs from the other groups (33.7 to 39.5 N). Furthermore, the eggshell strength of non-affected eggs in the NVC group was significantly lower (P<0.05) compared with that of non-affected eggs from the flock of origin (33.7 versus 41.2 N), but not different from the other groups. It can be concluded from the present study that vaccination with a live M. synoviae vaccine reduces the occurrence of M. synoviae-induced EAA significantly.

Experiments were performed (1) to quantify reflex movements and volume uptake of physiological salt solution by the cloaca and (2) to evaluate the conditioned cloacal uptake of fluorescein-labeled polystyrene. In experiment 1, measurements were done on birds (n = 12) once a day at 3, 4, and 5 d of age and during 5 consecutive days at wk 3, 5, 7, and 9 of age. The reflexes and volume uptake after applying saline droplets were studied simultaneously during 30 s. The median number and range of reflexes per 30 s during the first week of age were 45 (28 to 54), at 3 wk 35 (18 to 52), at 5 wk 44 (27 to 60), at 7 wk 47 (32 to 61), and at 9 wk 44 (23 to 56). The median volume uptake and range in wk 1, 3, 5, 7, and 9 were 0.10 (0.05 to 0.30), 0.25 (0.05 to 0.60), 0.58 (0.25 to 1.15), 1.05 (0.50 to 2.25), and 1.15 (0.30 to 3.05) mL per 30 s, increasing significantly with time. In experiment 2, a solution containing 10(7) polystyrene beads/mL was applied to the cloaca of broilers (3 aged 2 wk and 3 aged 9 wk) during 30 s. Most beads were found in the bursa of Fabricius. In the bursal lumen, a median of 10(6.43) beads/mL was found; the median number found in the follicular tissue was 5 (range 3 to 38) beads per tissue section. In the lumen content of ileum, cecum, and rectum of all birds together, it was 10(5.87), 0, and 10(6.32) beads/ ml, respectively. Polystyrene beads were never found intramuraly.

A novel eggshell pathology, characterized by an altered shell surface, thinning, increased translucency, and cracks and breaks in the eggshell apex, has become increasingly common in layer flocks of various breeds in The Netherlands. Two field studies found an association between the eggshell apex abnormalities (EAA) and infection with Mycoplasma synoviae. M. synoviae was isolated from the oviduct of birds that produced abnormal eggs, but not from birds in control flocks, although both affected and control birds had agglutinating antibodies against M. synoviae. After a single injection with long-acting oxytetracycline, the production of abnormal eggs ceased, but then reoccurred about 12 days later. A causal relationship between EAA and M. synoviae infection was subsequently demonstrated experimentally. EAA occurred after intratracheal inoculation of birds with M. synoviae, and even more frequently in birds infected with infectious bronchitis virus 5 days before inoculation of M. synoviae. EAA also occurred, although less frequently, in birds inoculated intravenously with M. synoviae and infected with infectious bronchitis virus. EAA did not occur in birds only inoculated intravenously with M. synoviae. M. synoviae was only isolated from the oviducts of birds producing abnormal eggs. The mean daily egg production was reduced in all groups infected with M. synoviae. Examination of the eggshells by scanning electron microscopy revealed that the mammillary layer of the calcified zone was absent and that the inner eggshell membranes were thicker. Isolates of M. synoviae from the oviducts of birds from farms producing abnormal eggs were examined by amplified fragment-length polymorphism analysis and did not appear to be clonal.

The progression of Escherichia coli lesions was studied in the respiratory tract of 4-week-old commercial broilers. Lesions were induced after a single intratracheal E. coli infection, and after an infection with E. coli preceded 5 days earlier by an oculo-nasal and intratracheal infectious bronchitis virus (IBV) infection of either the virulent M41 strain or the H120 vaccine strain. Trachea, lung and thoracic airsac lesions were examined macroscopically and microscopically. Tissue samples were taken at 3h post-inoculation (hpi), and 1, 2, 4 and 7 days post-inoculation (dpi) with E. coli. The location of both pathogens was assessed by immunohistochemistry. Single E. coli inoculation induced pneumonia and airsacculitis; in case it was preceded by IBV infection, the same macroscopical lesions and also viral tracheitis were found. No clear difference existed between the single and dual infected birds with respect to inflammatory reactions in the lung, which had disappeared within 7 days, except for the presence of more follicles in dual infected birds. IBV antigen was detected in secondary bronchi and airsacs up to 2 dpi and in the trachea up to 4 dpi. E. coli bacteria were found in the tracheal lumen included in purulent material, the parabronchi and airsacs. In lung tissue E. coli antigen was found up to 4 dpi. No clear difference existed between single and dual inoculated birds regarding the presence of E. coli in the lung. In the airsacs, a few bacteria were found from 0.5 hpi up to 4 dpi in E. coli and IBV-E. coli inoculated birds. Although both pathogens were cleared beyond detection at 7 dpi, in IBV-E. coli inoculated birds lesions in the airsac persisted, in contrast to broilers inoculated with E. coli only. In the present study it is shown that 4-week-old broilers are not resistant to intratracheal E. coli inoculation, however, these birds can overcome the induced E. coli infection within a short time span. Moreover, a preceding infection with vaccine or virulent IBV does not seem to impair the clearance of E. coli in the respiratory tract of broilers, but rather induces an exaggerated inflammatory response in the airsacs only, which seems to be the mechanism behind the pattern of airsacculitis in commercial poultry in the field.

Before the year 2000, Mycoplasma synoviae was associated mainly with subclinical respiratory infections in broilers in the Netherlands and was considered to have low clinical and economic impact. The subsequent occurrence of M. synoviae arthritis and amyloid arthropathy, and of eggshell apex abnormalities, has resulted in an increasing demand for M. synoviae-free poultry. Therefore, a cross-sectional seroprevalence study was carried out over a 12-month period during 2005 and 2006. Ten blood samples per farm were generally used because M. synoviae was expected to spread quickly. However, for grandparent and layer breeder stock, 24 to 60 blood samples per house were available from a voluntary M. synoviae monitoring programme. Sera were tested by means of the rapid plate agglutination test (agglutination at dilution > or =1:8 was considered positive). The numbers of farms sampled out of the national total were: broiler grandparent, 53/53; broiler parent rearing, 34/150; broiler parent, 114/300; broiler, 185/800; layer grandparent, 13/13; layer parent, 40/50; layer, 173/1250; and meat turkey, 50/75. The seroprevalence of M. synoviae in commercial poultry was high, especially in commercial layers where it was 73% (95% confidence interval (CI)=67 to 80); in layer and broiler grandparent stock, the seroprevalence was 0% and 10%, respectively, based on sample sizes equal to the population size. In layer and broiler parent farms, the seroprevalence was 25% (95% CI=19 to 31) and 35% (95% CI=28 to 44); in both broiler parent rearing and broiler farms it was 6% (95% CI=0 to 13 and 95% CI=3 to 9); and in meat turkey, the seroprevalence was 16% (95% CI=10 to 22).

Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.

Vaccination of chickens with dispersable dry powder vaccines was compared with commercial liquid vaccines. A Clone 30 Newcastle disease vaccine virus was spray dried with mannitol or with a mixture of trehalose, polyvinylpyrrolidone and bovine serum albumin. A coarse (+/-30 microm) and fine (+/-7 microm) powder were produced with both formulations. A commercial reconstituted Clone 30 vaccine was applied as coarse liquid spray (+/-222 microm) or fine liquid aerosol (+/-24 microm). Reduction of virus concentration in the air after dispersion/nebulization was monitored by air sampling and was explained by sedimentation of coarse particles/droplets and evaporation of fine droplets. The vaccine formulations induced high haemagglutination inhibition antibody titres in the serum of 4-week-old broilers (2(7) at 4 weeks post-vaccination). The good serum antibody response with the fine liquid aerosol despite extensive inactivation of virus due to evaporation of droplets, suggested that powder formulations (without inactivation due to evaporation) might allow a significant reduction of vaccine dose, thereby offering new options for fine aerosol vaccination with low-titre vaccines.

The in vitro susceptibility of 17 Dutch Mycoplasma synoviae isolates from commercial poultry to enrofloxacin, difloxacin, doxycycline, tylosin and tilmicosin was examined. Three isolates originated from joint lesions and 14 were from the respiratory tract. The type strain M. synoviae WVU 1853 was included as a control strain. Antibiotic susceptibility was tested quantitatively using the broth microdilution test. Based on initial and final minimum inhibitory concentration values, all tested isolates were susceptible to doxycycline, tylosin and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin and showed intermediate resistance to difloxacin.

The ban on chemotherapeutics against Histomonas meleagridis in the European Union has left turkey producers without an effective treatment against histomonosis. It has encouraged the development of alternative control methods amongst which are a number of herbal products. In the present study the in vitro effect of four herbal products against H. meleagridis was examined. After suspension of the herbal products in Dwyer's culture medium used for subculturing of a viable culture, parasite growth was monitored by frequently counting the histomonads until 72h of incubation. The solid products Aromabiotictrade mark, Enteroguardtrade mark, and Protophyt SPtrade mark as well as the positive control products dimetridazole and Histostat-50trade mark, were tested in twofold serial dilutions ranging in concentration from 400 to 12.5mug/mL, while the liquid product Protophyt Btrade mark was tested in concentrations ranging from 0.24% to 0.008% (v/v). The herbal products Enteroguardtrade mark and Protophyt Btrade mark as well as dimetridazole and Histostat-50trade mark, two chemotherapeutics with known antihistomonal activity, inhibited the growth of H. meleagridis, while no antihistomonal effect was found for Aromabiotictrade mark and Protophyt SPtrade mark.

During epidemics of classical swine fever (CSF), neighbourhood infections occurred where none of the 'traditional' routes of transmission like direct animal contact, swill feeding, transport contact or transmission by people could be identified. A hypothesized route of virus introduction for these herds was airborne transmission. In order to better understand this possible transmission route, we developed a method to detect and quantify classical swine fever virus (CSFV) in air samples using gelatine filters. The air samples were collected from CSFV-infected pigs after experimental aerosolization of the virus. Furthermore, we studied the viability of the virus with time in aerosolized state. Three strains of CSFV were aerosolized in an empty isolator and air samples were taken at different time intervals. The virus remained infective in aerosolized state for at least 30 min with half-life time values ranging from 4.5 to 15 min. During animal experiments, concentrations of 10(0.3)-10(1.6)TCID(50)/m(3) CSFV were detected in air samples originating from the air of the pig cages and 10(0.4)-10(4.0)TCID(50)/m(3) from the expired air of infected animals. This is the first study describing the isolation and quantification of CSFV from air samples originating from infected pigs and their cages, supporting previous findings that airborne transmission of CSF is feasible.

Histomoniasis is a serious disease in poultry. All chemotherapeutics with known efficacy against its causative agent, Histomonas meleagridis, have been banned from use as prophylactic or therapeutic use in production animals. In a search for possible alternatives, the in vivo effects of the herbal products Enteroguard and Protophyt were examined. Two-week-old turkeys allocated into 13 groups of 18 birds were either sham inoculated (negative control group) or were inoculated with 100, 3162 or 200 000 histomonads per bird. Control groups (no feed additives, dimetridazole, or Histostat-50) were included in the study. No morbidity or mortality was observed in the negative control group or in the groups inoculated with 100 histomonads per bird. Mortality was 100% in the groups inoculated with 200 000 histomonads per bird and either untreated (positive control group) or receiving Protophyt SP, Protophyt SP and Protophyt B, Enteroguard, or Histostat-50. Mortality was 17% in the dimetridazole-treated group. In the groups inoculated with 3162 histomonads per bird, mortality was 100% for the positive control group and the group receiving Enteroguard, and was 94% in the group receiving Protophyt SP. In the present study, Enteroguard or Protophyt was not found to be effective against histomoniasis.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Dwyer medium is the most frequently employed culture medium for Histomonas meleagridis. Both for subculturing and for resuscitation of H. meleagridis from storage in liquid nitrogen, modified Dwyer medium with an increased rice powder concentration (0.8%) and no chicken embryo extract proved superior to Dwyer standard medium, with threefold (10(6.3) vs. 10(5.8) histomonads/ml) to 10-fold (10(6.7) vs. 10(5.8) histomonads/ml) higher concentrations of parasites after resuscitation or subculturing, respectively.

A powder vaccine intended for aerosol vaccination of poultry was formulated by spray drying a live attenuated Newcastle disease virus with potential stabilizers (mannitol, trehalose, polyvinylpyrrollidone (PVP), bovine serum albumin (BSA)). Thermodynamic properties, water sorption, particle size distribution, nebulization properties, density and morphology of the powders were evaluated and the virus survival during spray drying and storage was determined by incubation in embryonated eggs and subsequent haemagglutination assay. All powders had a narrow size distribution with a median volume diameter of +/-30 microm (suitable for primary respiratory vaccination of chickens) and good aerosolization characteristics. Four amorphous, hygroscopic formulations were produced (trehalose, trehalose-PVP, trehalose-BSA, trehalose-PVP-BSA), where addition of BSA was beneficial for virus survival during production and storage at 6 and 25 degrees C. A crystalline, non-hygroscopic powder (mannitol) had a lower stabilizing capacity during production but maintained the remaining virus titre during storage. In conclusion, the study demonstrates that it is possible to produce a dry powder formulation of an attenuated live vaccine for mass vaccination of poultry in a one-step spray drying process.

As a part of the development of an efficient dry powder aerosol vaccine for poultry, the objective of this study was to accurately determine the deposition pattern of nebulized microspheres in the airways of unanaesthetized chickens of different ages (1 day, 2 weeks and 4 weeks old). In the first part of the study, the aerosol administration method was characterized: the influence of different nebulizers and nebulizing protocols on the relative humidity in the exposure chamber, the particle size distributions, the microsphere output and single microsphere percentage were determined. In the second part, birds were exposed to nebulized fluorescently labelled polystyrene microspheres (1 to 20 microm). Respiratory and gastro-intestinal tract tissue samples were collected and the number of fluorescent microspheres per sample was determined. In 2-week-old and 4-week-old chickens, microspheres of 5 and 10 microm, respectively, were too large for deposition in the lungs and air sacs as less than 5% of these microspheres penetrated into the lower airways. The larger size of microspheres reaching the lower airways of 4-week-old birds was explained by increasing airway dimensions with age. For 1-day-old chickens, deposition in the lungs decreased from 17 to 3% with increasing particle size (1 to 20 microm), but increased in the air sacs from 6 to 20%. Consequently, the total deposition percentage in the lower airways was independent of microsphere size and even 20 microm particles were able to penetrate into the lower airways, which was attributed to mouth breathing of the 1-day-old chickens.

E. coli bacteria isolated from localized and systemic disease processes in poultry are designated as Avian Pathogenic E. coli (APEC). The disease-inducing potential of these isolates has been explained by the occurrence of specific virulence factors. Despite the extensive literature on virulence factors for E. coli, unambiguous markers of virulence have not been identified yet. The relationship between serotyping and virulence is not straightforward either and raises the question whether E. coli infections in poultry should mainly be considered as opportunistic. Investigations into the occurrence of certain (combinations of) virulence factors in APEC isolates as virulence markers should fulfil the molecular version of Koch's postulates if the former question is to be answered.

Escherichia coli can induce salpingitis and/or peritonitis, a major cause of mortality in layer hens, but also other localized and systemic infections. E. coli infections have also been described in turkeys, geese, and ducks and are thought to be the cause of significant economic losses. However little is known about the real economic impact of the disease in layer chickens. The pathogenesis of E. coli salpingitis and peritonitis has not been elucidated yet. Three routes of infection have been discussed in the literature: ascending faecal contamination from the cloaca, bacterial translocation from the respiratory tract (air sac and lungs) and bacterial translocation from the intestinal lumen. Only one study has reported the occurrence of ascending faecal contamination from the cloaca to the oviduct and subsequently to the peritoneum. Regarding bacterial translocation, the only models available are for mammals, and these have not been applied to chickens so far Animal models could prove valuable to elucidate the pathogenesis of E. coli-induced salpingitis and peritonitis, and for assessing the value of preventive and curative intervention strategies. Little is known about risk factors for E. coli salpingitis and peritonitis. In contrast to colibacillosis in broilers, recent research has failed to demonstrate an association between several pathogens of the respiratory tract and the occurrence of E. coli pathology in layer chickens. The distance between poultry farms and the hen density in the cages were recently proposed as important risk factors for outbreaks ofcolibacillosis in flocks of layer hens, while in the past hormonal factors were implicated. The latter is an area of research that deserves more attention. Several methods for the molecular typing of E. coli have been described and might prove useful to study the epidemiology ofE. coli outbreaks in poultry, about which little is known. The presumptive diagnosis E. coli salpingitis and peritonitis is rather simple to establish, based on the anamnesis, clinical symptoms, and macroscopic findings at post-mortem. However; bacteriological analysis is required to establish a definite diagnosis because other pathogens can also cause salpingitis and peritonitis in layer hens. Antibiotics, chosen on the basis of sensitivity testing and their pharmacokinetic properties can be used as therapy; however residues in eggs may occur. Autovaccines are often used as prevention because in practice effective protection is only achieved against homologous E. coli serotypes.

The effect of a live Mycoplasma gallisepticum vaccine on the horizontal transmission of this Mycoplasma species was quantified in an experimental animal transmission model in specific pathogen free White Layers. Two identical trials were performed, each consisting of two experimental groups and one control group. The experimental groups each consisted of 20 birds 21 weeks of age, which were housed following a pair-wise design. One group was vaccinated twice with a commercially available live attenuated M. gallisepticum vaccine, while the other group was not vaccinated. Each pair of the experimental group consisted of a challenged chicken (10(4) colony-forming units intratracheally) and a susceptible in-contact bird. The control group consisted of 10 twice-vaccinated birds housed in pairs and five individually housed non-vaccinated birds. The infection was monitored by serology, culture and quantitative polymerase chain reaction. The vaccine strain and the challenge strain were distinguished by a specific polymerase chain reaction and by random amplified polymorphic DNA analysis. In both experiments, all non-vaccinated challenged chickens and their in-contact 'partners' became infected with M. gallisepticum. In the vaccinated challenged and corresponding in-contact birds, a total of 19 and 13 chickens, respectively, became infected with M. gallisepticum. Analysis of the M. gallisepticum shedding patterns showed a significant effect of vaccination on the shedding levels of the vaccinated in-contact chickens. Moreover, the Cox Proportional Hazard analysis indicated that the rate of M. gallisepticum transmission from challenged to in-contact birds in the vaccinated group was 0.356 times that of the non-vaccinated group. In addition, the overall estimate of R (the average number of secondary cases infected by one typical infectious case) of the vaccinated group (R = 4.3, 95% confidence interval = 1.6 to 49.9) was significantly lower than that of the non-vaccinated group (R = infinity, 95% confidence interval = 9.9 to infinity). However, the overall estimate of R in the vaccinated group still exceeded 1, which indicates that the effect of the vaccination on the horizontal transmission M. gallisepticum is insufficient to stop its spread under these experimental conditions.

Twenty European Eimeria spp. field isolates were subjected to an anticoccidial sensitivity test (AST). The anticoccidial drugs tested were diclazuril (Clinacox) and monensin (Elancoban). The assay was performed in a battery cage trial. Infected medicated birds were compared with an unmedicated control group. Coccidial lesion scores and oocyst shedding were used as parameters. The results of the AST show that resistance is common amongst coccidiosis field isolates, especially Eimeria acervulina (68% and 53% resistance for diclazuril and monensin, respectively). Resistance is less frequent amongst Eimeria maxima (38% and 50% resistance for diclazuril and monensin, respectively) and Eimeria tenella isolates (23% and 38% resistance for diclazuril and monensin, respectively). A highly significant influence of the coccidiosis prevention program (live coccidiosis vaccination with Paracox-5 vs. anticoccidial drugs in feed) on the sensitivity patterns of Eimeria spp. field isolates for both diclazuril (P= 0.000) and monensin (P= 0.001) was found. Further, when looking at the single species and each anticoccidial drug level, significantly more sensitivity of E. acervulina for monensin (P= 0.018), E. maxima for diclazuril (P = 0.009), and E. tenella for diclazuril (P = 0.007) was found in isolates originating from vaccinated flocks. Moreover, for E. acervulina and diclazuril, E. maxima and monensin, and E. tenella and monensin a trend toward higher sensitivity of isolates for these products was found when live coccidiosis vaccination was applied. The present study shows that sensitivity for the anticoccidial drugs diclazuril and monensin is more frequent in Eimeria spp. field isolates originating from broiler farms where a coccidiosis vaccination policy is followed.

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hens.

C-profiling is a novel genotyping method for protozoan pathogens, based on polymerase chain reaction and sequencing of AT-rich Internal Transcribed Spacer-1 sequences. It was applied to various Histomonas meleagridis isolates originating from outbreaks of histomoniasis in six Dutch turkey and chicken flocks. Three different H. meleagridis genotypes were identified. Type I and type II were associated with clinical disease. In two flocks, both having recovered from an outbreak of histomoniasis, a type III strain was found that was also morphologically slightly different from the type I and type II isolates. C-profiling is a promising technique to differentiate between H. meleagridis subtypes, making it useful for epidemiological studies.