Prevalence of netB-positive Clostridium perfringens in Italian poultry flocks by environmental sampling

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples ( n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.

Concurrent Occurrence of Lower Respiratory Aspergillosis and Pneumoconiosis in a Turkey Flock

This study describes a concurrent occurrence of lower respiratory aspergillosis and pneumoconiosis in a turkey flock. From one flock of 1000 turkeys, 4 clinically affected turkeys were examined pathologically and microbiologically. Clinically affected turkeys showed anorexia, lethargy, drooping of the wing, vomiting, dyspnea and open-beak breathing, or sudden dead. Gross lesions were observed in the air sacs and lungs which presented white to yellowish granulomas of varying size. Microscopic there was multifocal granulomatous airsacculitis and pneumonia. At the center of the granulomas, fungal hyphae were clearly highlighted in the lungs and air sacs by Gomori methenamie silver (GMS) staining. In the microbiological cultivation of the affected lungs and air sacs, Aspergillus fumigatus was isolated on Sabouraud dextrose agar. Furthermore, dark black dust accumulations were present in the cytoplasm of macrophages or interstitial tissue in the lungs and air sacs. There were also fibrosis and lymphohistiocytic cell infiltrations around the particles. Overall, this study could implicated that pneumoconiosis might be a predisposing factor in the development of respiratory aspergillosis in turkeys.

Micromorphological changes on the embryonic membranes of turkey eggs infected with Aspergillus fumigatus and their importance for embryonic survival

Aspergillosis is a frequent fungal disease of young and adult poultry in our commercial flocks. Infection can occur after hatching by inhalation of Aspergillus conidia which can be present in contaminated hatcheries, or later, by spores from moldy litter, dust, faeces or feed. Spores from the surface of egg shell can penetrate inside of an egg. The main characteristic of aspergillosis is granulomatous inflammation of respiratory system, although generalized form is possible as well. Multiple yellow nodules can be seen as major patomorphological changes and they are usually localized in lungs, air sacs, and can also be found in spleen, brain, subcutis and eyes. Egg embryos are quite susceptible to infection by Aspergillus fumigatus during incubation. In this study, the history of a case on one local farm with mortality rate of 7.2% in the turkey flock is presented. At the same time, 28 day old 30 incubated hatching turkey eggs were sampled, that were unable to hatch. The aim of the present work was to determine the cause and to identify the agent of embryonic mortality. Total of the 30 eggs were opened, and 16 of them had thickened egg membranes in the area of air sac. Membrane thickening was visible and circumscript or diffuse presence of black-grey or grey-green fungal growth was observed. Only 3 samples air sacs were filled with developed stages of fungi. To evaluate histopathological lesions, changed egg membranes were processed by standard histological technique. Dominant microscopic finding was thickening of egg membranes as a consequence of fungal growth and many of them penetrated deep towards embryo. Most of the hyphae were growing vertically through membranes. On the outside surface of the membranes, the elements of fungi (conidial heads with phialids and spores on them), could be clearly observed. These changes were responsible for embryonic death, which on the basis of the size of dead embryos occurred between 7th and 10th day of incubation. Aspergillus fumigatus was isolated and identified from the content of air sacs.

Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys

SUMMARYThe purpose of this study was to determine whether pooling avian influenza (AI)-positive swabs with negative swabs has a detrimental effect on the sensitivity of AI real-time reverse transcription–polymerase chain reactions (rRT–PCRs). Cloacal and buccal swabs were sampled daily from 12 turkeys infected with A/goose/England/07(H2N2). For half the turkeys, each swab was mixed with four swabs from known AI-negative turkeys, and for the other half the swabs were tested individually. Bayesian modelling was used to (i) determine whether pooling the positive swabs compromised the cycle threshold (Ct) value obtained from the rRT–PCRs, and (ii) estimate the likelihood of detection of an H2N2 infected turkey flock via rRT–PCR for pooled and individually tested swabs (cloacal and buccal)vs. the number of days post-infection of the flock. Results indicated that there was no significant effect of compromising AI rRT–PCR sensitivity by pooling a weak positive swab with negative swabs on the Ctvalues which were obtained. Pooled sampling was able to widen the detection window compared to individual sampling, for the same number of rRT–PCR tests. This indicates that pooled sampling would be an effective method of reducing the number of tests to be performed to determine flock status during an AI outbreak and for surveillance.