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Ornithobacterium rhinotracheale is an avian respiratory pathogen that affects turkeys. The objective of this study was to evaluate the clinical efficacy of gamithromycin (GAM) against O. rhinotracheale in turkeys. The birds were inoculated oculonasally with 10(8) colony-forming units (cfu) of O. rhinotracheale, preceded by infection with avian metapneumovirus. In addition to a negative (CONTR-) and a positive control group (CONTR+) there were two treated groups administered GAM (6 mg/kg) either subcutaneously (GAM SC) or orally (GAM PO) by administration as a single bolus at one-day post-bacterial infection (p.b.i.). From the start of the avian metapneumovirus infection until the end of the experiment, the turkeys were examined clinically and scored daily. In addition, tracheal swabs were collected at several days p.b.i. Necropsy was performed at 4, 8 and 12 days p.b.i. to evaluate the presence of gross lesions, and to collect trachea and lung tissue samples and air sac swabs for O. rhinotracheale quantification. The clinical score of the GAM SC group showed slightly lower values and birds recovered earlier than those in the GAM PO and CONTR+ groups. O. rhinotracheale cfus were significantly reduced in tracheal swabs of the SC group between 2 and 4 days p.b.i. At necropsy, CONTR+ showed higher O. rhinotracheale cfu in lung tissues compared to the treated groups. Moreover, at 8 days p.b.i. only the lung samples of CONTR+ were positive. In conclusion, the efficacy of GAM against O. rhinotracheale was demonstrated, especially in the lung tissue. However, the PO bolus administration of the commercially available product was not as efficacious as the SC bolus.

Two 1-year old Roulroul partridges (Rollulus rouloul), one male and one female, were presented because of eye problems and anorexia. Twenty of the 30 Roulroul partridges in the owner's collection had already died. The affected birds stopped eating, became thinner, and eventually died. Antibiotic treatment, which started because of the suspicion of a septicaemic process, was unsuccessful. At clinical examination of the two partridges it was found that in both birds, one eye ball was filled with a whitish yellow amorphous material and the other eye ball of the female showed a distinct corneal opacity. Both presented birds were euthanized. Necropsy revealed no significant abnormalities in addition to the eye lesions. Histology and immunohistochemistry of the female's eye revealed an infiltrate of T-lymphocytes corresponding to ocular lymphoma. Herpesvirus genus-specific PCR, followed by Sanger sequencing confirmed the presumptive diagnosis of Marek's disease in both birds. To our knowledge, this is the first confirmed case of infection with Gallid Herpesvirus 2 (Marek's disease virus) in partridges and the first case in this specific species.

In poultry rearing, medicated drinking water is a commonly used administration route, but drug uptake can be affected by many factors. In this study, the influence of two important parameters, the photoperiod and feeding schemes, on florfenicol uptake in turkeys was tested. First, the uptake was determined as the pharmacokinetic/pharmacodynamic profile of florfenicol; and second, we evaluated the clinical efficacy of florfenicol against Ornithobacterium rhinotracheale. Both experiments were conducted during a 5-day treatment of 30 mg/kg body weight florfenicol administered via drinking water and considering different photoperiods and feeding schemes (group 20/4L: photoperiod of 20 h, fed ad libitum; group 16/8L: photoperiod of 16 h, fed ad libitum; group 16/8R: photoperiod of 16 h, fed ad libitum but feed was withdrawn during the dark period and replaced 1 h after lighting). On day 1 of treatment, all groups showed plasma concentrations above the minimum inhibitory concentration (both MIC50 and MIC90, 1 mg/l) of 37.7%, 63.5% and 53.1% of a 24-h interval for 20/4L, 16/8L and 16/8R, respectively. Only in the 16/8L and 16/8R groups was the MIC also exceeded on day 5 (47.9% and 21.5% of a 24-h interval, respectively). In all groups, a clinical improvement could be noticed, resulting in reduction of the clinical score. However, only the 16/8L and 16/8R groups showed significant differences from the control group. The results demonstrated an important influence of the photoperiod on the pharmacokinetics of florfenicol as well as the clinical outcome in an infection model. It can be advised that the photoperiod should be <20 h to have sufficient drug intake. Nevertheless, there was no effect between fed and fasted turkeys for both the pharmacokinetics and the clinical outcome.

Aspergillosis is the most common fungal disease of the avian respiratory tract and is caused primarily by Aspergillus fumigatus. The respiratory macrophages provide important defence against aspergillosis. T-2 toxin (T-2), a trichothecene mycotoxin produced by Fusarium spp. in improperly stored agricultural products, has immunomodulatory effects. We studied the impact of T-2 on the antifungal response of the chicken macrophage cell line HD-11 against A. fumigatus infection. The macrophages were first exposed to 0.5 to 10 ng/ml T-2 for 24 h, and then their viability, antifungal activity, and cytokine expression in response to A. fumigatus conidial infection were determined. The viability of macrophages decreased when exposed to T-2 at concentrations higher than 1 ng/ml. One hour after conidial infection, phagocytosed conidia were observed in 30% of the non-T-2-exposed macrophages, but in only 5% of the macrophages exposed to 5 ng/ml T-2. Seven hours after infection, 24% of the conidia associated with non-T-2-exposed macrophages germinated, in contrast to 75% of those with macrophages exposed to 5 ng/ml T-2. A. fumigatus infection induced upregulation of interleukin (IL)-1β, CXCLi1, CXCLi2 and IL-12β, and downregulation of transforming growth factor-β4 in macrophages. Exposure of A. fumigatus-infected macrophages to T-2 at 1 to 5 ng/ml further upregulated the expression of IL-1β, IL-6, CCLi2, CXCLi1, CXCLi2, IL-18 (at 1 and 2 ng/ml) and IL-12β, and further downregulated that of transforming growth factor-β4 (at 5 ng/ml). In conclusion, T-2 impaired the antifungal activities of chicken macrophages against A. fumigatus conidia, but might stimulate immune response by upregulating the expression of pro-inflammatory cytokines, chemokines and T-helper 1 cytokines.

Aspergillosis caused by Aspergillus fumigatus seems to be more prevalent in some avian species than in others. We compared the development of aspergillosis in 8-month-old Gyr-Saker hybrid falcons and 8-month-old pigeons after a single intratracheal inoculation of different dosages of A. fumigatus conidia (10(7), 10(5) and 10(3)). Clinical signs, including vomiting, discoloration of the urates, loss of appetite and dyspnoea, were observed in four out of five falcons and in four out of five pigeons inoculated with 10(7) A. fumigatus conidia. Necropsy revealed the presence of granulomas in the air sacs and/or lungs in four out of five falcons and in four out of five pigeons in the high dosage group. A. fumigatus was isolated from these granulomas in three falcons and in three pigeons. The presence of fungal hyphae was detected with Periodic acid Shiff reagent staining in three out of five falcons and in three out of five pigeons in the high dosage group. Avian respiratory macrophages were clearly present in and around the fungal granulomas. In the other dosage groups, no granulomas, positive A. fumigatus cultures or fungal hyphae were present, except for one falcon in the middle dosage group in which a sterile granuloma without fungal hyphae was noticed. In conclusion, the study shows that adult falcons and pigeons are susceptible to aspergillosis after inoculation of a single dose of conidia intratracheally.

Aspergillosis is one of the most common causes of death in captive birds. Aspergillosis in birds is mainly caused by Aspergillus fumigatus, a ubiquitous and opportunistic saprophyte. Currently it is not known whether there is a link between the environmental isolates and/or human isolates of A. fumigatus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyse 65 clinical avian isolates and 23 environmental isolates of A. fumigatus. The 78 genotypes that were obtained were compared with a database containing genotypes of 2514 isolates from human clinical samples and from the environment. There appeared to be no specific association between the observed genotypes and the origin of the isolates (environment, human or bird). Eight genotypes obtained from isolates of diseased birds were also found in human clinical samples. These results indicate that avian isolates of A. fumigatus may cause infection in humans.

Aspergillosis is an infectious, non-contagious fungal disease caused by species in the ubiquitous opportunistic saprophytic genus Aspergillus, in particular Aspergillus fumigatus. This mycosis was described many years ago, but continues to be a major cause of mortality in captive birds and, less frequently, in free-living birds. Although aspergillosis is predominantly a disease of the respiratory tract, all organs can be involved, leading to a variety of manifestations ranging from acute to chronic infections. It is believed that impaired immunity and the inhalation of a considerable amount of spores are important causative factors. The pathogenesis, early diagnostic methods and antifungal treatment schedules need to be further studied in order to control this disease. The aim of the present review is to present the current knowledge on aspergillosis with the main emphasis on A. fumigatus infections in captive and free-living birds rather than domestic poultry. The review covers aetiology, epidemiology, pathogenesis, clinical signs and lesions, diagnosis, treatment and prevention.

Drinking-water treatment with enrofloxacin is widely used to cure respiratory infections in turkeys. The current treatment regimen advises a 5-day treatment at 10 mg/kg body weight. Since enrofloxacin exerts a concentration-dependent activity it might be useful to provide the total treatment dose of 50 mg/kg total dose in a single-day treatment regimen. We therefore assessed whether single-day treatment regimens with 50 mg/kg body weight were clinically equivalent to the advised multiple-day treatment regimen with 10 mg/kg body weight for 5 days. For this purpose, five groups of 16 turkeys, 22 days old, were experimentally inoculated with avian metapneumovirus (APV) and Ornithobacterium rhinotracheale and subsequently treated in the drinking water with enrofloxacin, using either a single-day treatment regimen at 50 mg/kg body weight during a 5-h, 10-h or 20-h period or a standard 5-day treatment regimen at 10 mg/kg body weight/ day for 20 h. Although initially all dosage regimens cleared O. rhinotracheale from the trachea, 4 days after onset of treatment O. rhinotracheale bacteria were re-excreted in the single-day regimens but without worsening of the clinical symptoms. The 5-day treatment with 10 mg enrofloxacin/kg in turkeys provided the best results for the treatment of an O. rhinotracheale infection in turkeys by shortening the course and reducing the severity of clinical disease and by eliminating O. rhinotracheale from the respiratory tract without re-emergence. None of the used treatment regimens promoted the selection of bacterial clones with reduced susceptibility or resistance.

In vivo modelling of aspergillosis in birds allows the evaluation of control measures and the study of host-pathogen interactions. In this study the impact of the use of different inoculation routes and immunosuppression on the course of an infection with Aspergillus fumigatus in racing pigeons (Columba livia domestica) was examined. A. fumigatus conidia were inoculated in the thoracic air sac, lung or trachea in immunocompetent or immunosuppressed pigeon squabs. Immunosuppression was induced by three dexamethasone injections before inoculation. Mortality in the A. fumigatus-inoculated groups varied between 1/4 and 4/4. The highest and more acute mortality was seen in immunocompetent pigeons inoculated in the thoracic air sac and in pigeons inoculated in the thoracic air sac or lung after immunosuppression. Pigeons inoculated in the lung or inoculated intratracheally after immunosuppression developed an aspergillosis infection with a slower course of disease and more prominent clinical symptoms. Using microsatellite length polymorphism, it was confirmed that all mycoses were caused by the inoculated strain except for one isolate in a dexamethasone-treated pigeon. In conclusion, inoculation in the lung is selected as the preferred model for chronic aspergillosis in pigeons, and inoculation in the thoracic air sac as the preferred model for acute aspergillosis. The use of immunosuppressed birds seems to be contra-indicated due to the risk of opportunistic infections.

The usefulness of butyrate, acetate, propionate and l-lactate for the control of Campylobacter jejuni infections in broilers was assessed. For this purpose, the effect of these acids on the growth of C. jejuni in broth and intestinal mucous was determined, as well as their influence on the invasiveness of C. jejuni in intestinal epithelial cells. From these in vitro obtained results, one acid was retained for use as a feed additive in an in vivo trial. Butyrate was the most successful of the short-chain fatty acids, with 12.5 mM being bactericidal for C. jejuni at pH 6.0. Propionate and acetate had a bacteriostatic effect at 50 mM. None of the short-chain fatty acids had a bactericidal effect at pH 7.5 at a maximum concentration of 50 mM. Mucous increased the minimum bactericidal concentration of butyrate, but not the bacteriostatic concentrations of propionate or acetate. When C. jejuni was incubated in growth subinhibitory concentrations of butyrate, acetate or propionate or 25 mM L-lactate, no alteration in the invasive capabilities of C. jejuni in Caco-2 cells was observed. The addition of butyrate-coated micro-beads to the feed was unsuccessful to reduce C. jejuni caecal colonization in a seeder model using 2-week-old broilers. In conclusion, despite the marked bactericidal effect of butyrate towards C. jejuni in vitro, butyrate-coated micro-beads do not protect broilers from caecal colonization with C. jejuni in the applied test conditions. This might be partially ascribed to the protective effect of mucous and the rapid absorption of butyrate by the enterocytes.

Salmonella enterica serotype Enteritidis is the predominant serotype associated with egg-borne salmonellosis in humans. Apparently this serotype possesses particular characteristics that increase its chance to contaminate eggs. To identify these characteristics, two Salmonella serotype Enteritidis strains as well as one strain of each of the serotypes Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Virchow and Salmonella Hadar strain were used to examine different aspects related to egg contamination. After an intravenous infection of laying hens, it was observed that the ability of the serotype Enteritidis strains to colonize the reproductive organs was significantly higher compared with the Salmonella Heidelberg, Salmonella Virchow and Salmonella Hadar strains but not with the Salmonella Typhimurium strain. Inoculating low numbers of the different Salmonella serotypes in egg albumen at 42 degrees C demonstrated that the growth of the strains belonging to the Salmonella serotypes Virchow and Hadar was seriously repressed. The other serotypes, however, survived in albumen for 24 h. Furthermore, using two different specifically designed egg infection models, it was shown that all strains used in this study were able to penetrate into and multiply inside the yolk at 25 degrees C. These findings indicate that the ability to grow in eggs post lay is not specific for the serotype Enteritidis. In conclusion, comparing strains belonging to different Salmonella serotypes has revealed that most probably a preferential colonization of the reproductive organs and an enhanced survival at 42 degrees C allows the serotype Enteritidis to contaminate eggs.

Currently Clostridium perfringens-induced necrotic enteritis is a major problem in broiler flocks. In the present study, broilers were inoculated with a combination of Eimeria maxima or overdose coccidial vaccine (one inoculation) with C. perfringens (repeated inoculations). Single C. perfringens, E. maxima or an overdose of live coccidial vaccine inoculations did not result in grossly visible necrotic gut lesions, while combined inoculation resulted in typical necrotic lesions at approximately 4 days after inoculations with C. perfringens in approximately one-half of the inoculated animals. Semi-quantitative histological lesion scoring was done to evaluate gut damage in gut sections of animals in which no gross necrotic lesions were detected. This included scoring of hyperaemia, haemorrhages, the amount of red blood cells and protein precipitate in the lumen, villus fusion and epithelial defects. The villus length and the villus length/crypt depth ratio were also analysed. This approach proved to be discriminative between single E. maxima infection, overdose of coccidial vaccine or C. perfringens inoculations and the non-inoculated control group, and between the double-inoculated and single-inoculated groups. In general, the highest histological scores for gut lesions were observed in the double-inoculated groups, but the single-inoculated groups had higher scores than the control group. It was concluded that oral inoculation of broilers with an overdose of live coccidial vaccine in combination with multiple oral C. perfringens inoculations is a suitable model for necrotic enteritis without inducing mortality of the animals. C. perfringens and Eimeria act synergistically in inducing grossly visible gut damage.

A total of 73 isolates of Enterococcus spp. carrying the erm(B) gene were obtained from cloacal swabs of broiler chickens derived from 13 different farms in Belgium. The erm(B) gene encodes resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS(B)). The isolates belonged to eight different species: Enterococcus avium (eight isolates), Enterococcus casseliflavus (11 isolates), Enterococcus cecorum (eight isolates), Enterococcus durans (seven isolates), Enterococcus faecalis (10 isolates), Enterococcus faecium (17 isolates), Enterococcus gallinarum (seven isolates) and Enterococcus hirae (five isolates). Acquired resistance to tetracycline was detected in 68 of the isolates, and in 62 of these it was associated with the presence of the resistance genes tet(L), tet(M), tet(O) or tet(S). In three E. faecium isolates that were phenotypically susceptible to tetracycline, tet(L) or tet(M) was present. The transposon integrase gene (int gene) of the Tn916/Tn1545 transposon family was detected in 18 of the 54 isolates that contained the tet(M) gene. It was concluded that acquired resistance to tetracycline antibiotics is often present in enterococci from poultry carrying the erm(B) gene. The use of tetracyclines in poultry may therefore co-select for resistance to MLS(B) antibiotics, which may be important as alternative therapy for enterococcal infections in humans.

Salmonella is a human pathogen that is commonly found in poultry products. It is possible to decrease chicken carcass and egg contaminations by adding organic acids to the feed or drinking water at appropriate times. Medium-chain fatty acids are more antibacterial against Salmonella than short-chain fatty acids. The antibacterial effect of these acids is species specific. Bacteria that are unable to decrease intracellular pH accumulate organic acid anions in accordance with the pH gradient across their cell membranes. The short-chain fatty acid butyrate specifically down-regulates expression of invasion genes in Salmonella spp. at low doses. Also medium-chain fatty acids and propionate decrease the ability of Salmonella spp. to invade epithelial cells, in contrast to acetic acid. Because not all bacteria are affected in a similar fashion by organic acids, it may be possible to use probiotic and prebiotic bacteria to achieve beneficial effects. If diets can be designed to stimulate organic acid production in the caecum, it may be possible to control Salmonella spp. via even easier and more cost-effective measures, compared with addition of acids to feed or drinking water.

Eighty-seven Lactobacillus strains isolated from cloacal swabs of broiler chickens derived from 20 different farms in Belgium were identified to species level and tested for susceptibility to macrolide and lincosamide antibiotics. Five different Lactobacillus species were identified as being predominantly present in the cloacae of broilers: Lactobacillus crispatus, Lactobacillus salivarius subsp. salivarius, Lactobacillus amylovorus, Lactobacillus gallinarum and Lactobacillu sreuteri. Acquired resistance prevalence to macrolides and lincosamides was very high in the investigated lactobacilli: 89% of the strains were resistant to either or both lincosamide and macrolide class antibiotics. The vast majority of these resistant strains (96%) displayed constitutive resistance. More than one-half of the macrolide and/or lincosamide resistant strains carried an erm(B), erm(C), mef(A), lnu(A) gene or a combination of these genes.

The incidence of Clostridium perfringens-associated necrotic enteritis in poultry has increased in countries that stopped using antibiotic growth promoters. Necrotic enteritis and the subclinical form of C. perfringens infection in poultry are caused by C. perfringens type A, producing the alpha toxin, and to a lesser extent type C, producing both alpha toxin and beta toxin. Some strains of C. perfringens type A produce an enterotoxin at the moment of sporulation and are responsible for foodborne disease in humans. The mechanisms of colonization of the avian small intestinal tract and the factors involved in toxin production are largely unknown. It is generally accepted, however, that predisposing factors are required for these bacteria to colonize and cause disease in poultry. The best known predisposing factor is mucosal damage, caused by coccidiosis. Diets with high levels of indigestible, water-soluble non-starch polysaccharides, known to increase the viscosity of the intestinal contents, also predispose to necrotic enteritis. Standardized models are being developed for the reproduction of colonization of poultry by C. perfringens and the C. perfringens-associated necrotic enteritis. One such model is a combined infection with Eimeria species and C. perfringens. Few tools and strategies are available for prevention and control of C. perfringens in poultry. Vaccination against the pathogen and the use of probiotic and prebiotic products has been suggested, but are not available for practical use in the field at the present time. The most cost-effective control will probably be achieved by balancing the composition of the feed.

Colibacillosis appears to be of increasing significance in layer flocks, but there have been no studies of the risk factors associated with outbreaks. This study aimed to investigate the possible associations between risk factors of non-infectious nature and outbreaks of mortality due to colibacillosis in flocks of caged layer hens. Information on management, biosecurity measures and housing conditions was collected in 20 flocks suffering from the disease and in 20 clinically healthy control flocks. The data were processed using multiple logistic regression. The statistical analysis demonstrated that an increase in the distance to the nearest poultry farm by 1 km was associated with a six-fold decreased risk of an outbreak of colibacillosis (odds ratio=0.16). Furthermore, a 1 l increase in cage volume per hen was associated with a 33% decrease in the risk of an outbreak (odds ratio=0.75). It was concluded that the distance between poultry farms and the hen density in the cages are important risk factors for outbreaks of colibacillosis in flocks of layer hens.

This study aimed to examine the significance of interactions between Escherichia coli and various respiratory pathogens during outbreaks of colibacillosis-associated mortality in layer hen flocks under field conditions. For this purpose, a case-control study involving 20 control flocks with baseline mortality and 20 flocks with increased mortality due to E. coli septicaemia and polyserositis, was conducted. In each colibacillosis flock, blood samples were taken from 20 hens at the onset of clinical disease and three times thereafter at 2-week intervals. Control flocks of comparable ages were sampled in the same way. Pooled sera, taken at the first and last sampling, were examined for antibody titres against infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), and the individual sera from all four samplings were examined for the presence and/or titres of antibodies against avian pneumovirus (APV), Mycoplasma gallisepticum, Mycoplasma synoviae and Ornithobacterium rhinotracheale. Titre increases were seen for IBV D274 (one control flock) and O. rhinotracheale (one control and one colibacillosis flock). An increase in per cent reactors was seen for APV (one control flock), and for M. synoviae (one control and two colibacillosis flocks). The study failed to detect any consistent interactions between E. coli and the aforementioned pathogens. These results indicate that, at least as observed in this study, outbreaks of increased mortality resulting from colibacillosis are not necessarily associated with IBV, NDV, APV, M. gallisepticum, M. synoviae or O. rhinotracheale infections.

In Europe, outbreaks of acute mortality in layer flocks due to colisepticaemia have frequently been observed since the mid-1990s. The aims of this study were to describe the disease, to identify the serotypes of the avian pathogenic Escherichia coli (APEC) present in these outbreaks, and to detect the presence of F11 fimbriae and flagella in the isolates. For this purpose, 20 flocks with APEC-associated increased mortality and 20 control flocks matched for age were examined. Weekly mortality rates in the colibacillosis-affected flocks reached 1.71%, versus 0.30% in the control flocks. The maximum cumulative mortality over an entire colibacillosis outbreak reached 9.19%. The disease was often flock and hen house associated, with recurrent outbreaks within one round and in successive rounds in the same house. Disease was usually acute without clinical symptoms. Peritonitis with yolk material deposited in the peritoneal cavity and polyserositis were the main lesions at necropsy. O78 strains were isolated in 15 of the 20 colibacillosis flocks, and in only one of the control flocks. The majority of strains from the control flocks could not be serotyped by the 28 O-antisera used. In general, F11 fimbriae and flagella were present in the majority of the strains. F11 fimbriae were significantly more often found in O78 isolates than in the other serotypes, and are thus more often present in isolates from colibacillosis flocks. Strains positive for F11, and for F11 and flagella, were more frequently present in heart and liver of the colibacillosis-affected flocks.

Although Cryptococcus laurentii has been isolated from fresh droppings and cloaca samples from feral pigeons, it has never before been associated with an actual disease condition in birds. This case study deals with the first report on C. laurentii associated with feather loss in a glossy starling (Lamprotornis chalybaeus). The bird exhibited patchy feather loss, especially around the back and beak area, and greyish crusts sticking quite firmly to the underlying skin. The feathers had a greasy appearance and disseminated a musty odour. Treatment was installed with fluconazole in the drinking water. One month following the onset of treatment, the condition of the plumage had markedly improved.