Biodistribution of 89Zr-DFO-labeled avian pathogenic Escherichia coli outer membrane vesicles by PET imaging in chickens.

Avian pathogenic Escherichia coli (APEC) is a serious systemic infectious disease in poultry infections, causing severe economic losses to the poultry industry. Previous studies have shown that secretion of virulence proteins was required for the pathogenicity of APEC through the secretion system. Outer membrane vesicles (OMVs) are a generalized secretion system of Gram-negative bacteria that play a key role in the long-distance delivery of virulence factors, but whether they are associated with the pathogenic mechanism of APEC has not been determined. In this study, OMVs were purified and characterized from AE17 (O2 serotype) by ultracentrifugation and density gradient centrifugation and their protein cargo was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, 89Zr was labeled after chelating AE17 OMVs by DFO and positron emission tomography PET imaging was used to track 89Zr-DFO-OMVs in chickens and to pathologically analyze the distribution sites. This study showed that AE17 OMVs were membrane vesicles ranging in size from 20 to 200 nm and proteomic analysis revealed the presence of virulence proteins, including adhesion proteins OmpA, OmpC, OmpF, OmpX, FimH, FimC and FigE, and serum resistance proteins OmpT and MliC and immune response regulator proteins (FliC). In addition, in vivo PET imaging to track the biodistribution of AE17 OMVs showed that AE17 OMVs were taken up by the lung region and the gastrointestinal and renal regions but were not detected in other areas. Pathological analysis of the tissue sites where AE17 OMVs were ingested showed inflammatory responses and damage. These findings suggested that AE17 OMVs not only contained a group of virulence proteins associated with AE17 infection but can also deliver these virulence proteins over long distances and caused tissue inflammatory damage. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of APEC that could aid in the development of vaccines and antibiotics effective against APEC.

Objective scoring of footpad dermatitis in broiler chickens using image segmentation and a deep learning approach: camera-based scoring system.

1. Footpad dermatitis (FPD) can be used as an important indicator of animal welfare and for economic evaluation; however, human scoring is subjective, biased and labour intensive. This paper proposes a novel deep learning approach that can automatically determine the severity of FPD based on images of chicken's feet.2. This approach first determined the areas of the FPD lesion, normal parts of each foot and the background, using a deep segmentation model. The proportion of the FPD for the chicken's two feet was calculated by dividing the number of FPD pixels by the number of feet pixels. The proportion was then categorised using a five-point score for FPD. The approach was evaluated from 244 images of the left and right footpads using five-fold cross-validation. These images were collected at a commercial slaughter plant and scored by trained observers.3. The result showed that this approach achieved an overall accuracy and a macro F1-score of 0.82. The per-class F1-scores from all FPD scores (scores 0 to 4) were similar (0.85, 0.80, 0,80, 0,80, and 0.87, respectively), which demonstrated that this approach performed equally well for all classes of scores.4. The results suggested that image segmentation and a deep learning approach can be used to automate the process of scoring FPD based on chicken foot images, which can help to minimise the subjective bias inherent in manual scoring.

Reproductive tract diseases in female backyard chickens (Gallus gallus domesticus) - diagnostic imaging and final outcome during a decade.

OBJECTIVE: Female reproductive tract disorders are common conditions of backyard poultry with an increasing demand for individual veterinary care. However, only limited case reports are available on diagnostic workup and outcomes of individual cases. This study aims at giving an overview of usually presented reproductive tract disorders, comparing diagnostic imaging findings with final diagnoses, and summarizing the outcome of the respective diseases. MATERIAL AND METHODS: The digital medical records archive of the University for Veterinary Medicine in Vienna was searched for chickens that were finally diagnosed with diseases of the reproductive tract, including all patients from May 1st, 2009 to May 31st, 2019. Information such as patient age, medical history, results of diagnostic imaging, final confirmed diagnosis, outcome, surgical protocol and necropsy findings was extracted. RESULTS: Finally confirmed reproductive tract diseases were found in 57 of 315 female chickens. The most common conditions were egg-related coelomitis along with salpingitis or impacted salpinx (25/57), followed by ovarian or oviductal neoplasia (17/57). Clinical findings were unspecific in the majority of cases, but most conspicuous were chickens presented with a distended coelomic cavity and apathy. Coelomic ultrasonography as well as computed tomography proved to be valuable tools for distinguishing between the respective conditions. However, ultrasonography alone mainly failed (10/11) to differentiate between ovary or oviductal neoplasia and egg-related coelomitis with salpingitis or impacted salpinx, respectively. Computed tomography was perceived as a superior tool for final diagnosis. In total 6/6 CT-scans correctly made a definitive diagnosis. Nevertheless, accurate diagnosis was only possible after celiotomy with the necessity of consecutive surgery for most of the reported cases. As an overall outcome 34 of 57 patients were either euthanised or died, whereas only 23 chickens could be successfully treated and discharged. Several hens were reported to be doing well at home, according to regular check-up procedures within 4 years post-surgery. CONCLUSION AND CLINICAL RELEVANCE: Diagnostic work-up and treatment of hens with reproductive tract diseases can be challenging. Clinical presentation and diagnostic imaging provide important information, still celiotomy is often required for final diagnosis. The condition of the chickens is usually serious. Within our study, 40 % of the hens could be treated successfully. Therefore, a realistic assessment of each individual case and clarification for the owners are important.

Avian pulmonary proteinosis: six cases and a review of the literature.

Pulmonary alveolar proteinosis (PAP) is a disease of surfactant clearance in which functional abnormalities in alveolar macrophages lead to accumulation of surfactant within alveoli in mammals. Histologic examination of 6 avian autopsies, including 4 chickens, a turkey, and a cockatiel, revealed accumulation of hypereosinophilic densely arrayed lamellar material in the lungs that was magenta by periodic acid-Schiff stain and diastase resistant. Transmission electron microscopy of the proteinaceous material in 2 cases demonstrated alternating electron-dense and electron-lucent lamellae that formed whorls and had a regular periodicity of 6-14 nm, consistent with pulmonary surfactant. Given the anatomic differences between avian and mammalian lungs, we designated the presented condition "pulmonary proteinosis," which can be observed as both an incidental finding or, when severe, may be a contributing factor to death through respiratory failure.

CT venography and pathological characterization of crus haemorrhage in Pekin ducks.

The crus haemorrhage is one of the main causes of carcass defects in Pekin duck processing houses. However, its pathologic features are currently unclear. In order to examine the injury to the hind limb veins and illustrate the pathologic characteristics of crus haemorrhage in Pekin ducks, a total of 68 Pekin ducks with crus haemorrhage (test group) and 10 unaffected ducks (control group) were collected in this study. Five ducks randomly selected from each group were examined by computed tomographic venography with 2.0 mm thickness, 120 kVp, and 90 mA. Pathological changes were observed macroscopically, and under a microscope and electron microscope. The computed tomographic venography results showed no differences in the main hind limb veins between Pekin ducks with crus haemorrhage and the control. Macroscopic results demonstrated that the haemorrhage only occurred in crural muscles, most frequently in musculus gastrocnemius and musculus tibialis cranialis. In severe cases, muscular rupture and multiple intermuscular blood clots could be observed. Histological analysis showed rupture of myofibers and massive red blood cells between muscle bundles. Besides, infiltration of connective tissues and inflammatory lesions could be seen. However, no differences were observed in other organs between these two groups. The main ultrastructural characteristics were myofibrillar rupture and split, accompanied by mitochondrial membrane disintegration and vacuolization. All these results indicate that the haemorrhage in crus is a focal myopathy with the characteristics of bleeding, rupture, and inflammatory lesions. Research highlights CTV was a feasible method to evaluate the hind limb veins in Pekin ducks. The focal myopathy presented here only affected crural muscles. The focal myopathy was characterized by bleeding, rupture and inflammatory lesions.

Radiography and Ultrasonography in the Backyard Poultry and Waterfowl Patient.

The keeping of backyard poultry and waterfowl as pets has become increasingly popular in recent years, resulting in a rising case load of these patients in veterinary practices. Diagnostic imaging techniques are taking a leading role in rapid diagnosis in the live bird. We provide an overview of the most important points regarding radiographic and ultrasonographic imaging procedures in these birds. We also review the most commonly documented radiographic and ultrasonographic signs in these species, as well as discuss unique anatomic characteristics with which the veterinarian should be familiar.

Development of a TaqMan-based real-time PCR assay for rapid and specific detection of fowl aviadenovirus serotype 4.

Twelve serotypes of fowl aviadenovirus, namely, FAdV-(1-8a and 8b-11), have been identified, among which FAdV-4 is the aetiologic agent of hepatitis hydropericardium syndrome (HHS) in chickens. Outbreaks of HHS have been documented in many countries, causing significant economic losses. Real-time PCR methods described so far in the literature cross-detect different serotypes of FAdVs. In this study, we aimed to develop a TaqMan-based real-time PCR assay for the specific detection of FAdV-4. A pair of primers targeting the hexon gene and a TaqMan probe were designed. Using different copy numbers of plasmid DNA carrying the hexon gene as template, we showed the detection limit of this assay was 101 copies/reaction, which was 10 times higher than conventional PCR. The assay was highly specific for FAdV-4 and did not cross-detect 11 other serotypes of FAdVs, avian influenza virus, Newcastle disease virus, infectious bronchitis virus or subgroup J of the avian leukosis virus. The reproducibility of the assay was assessed by five independent reactions using different copy numbers of plasmid DNA (103 and 105) as template, and the results showed 0.56-1.15% coefficient of variation for inter-assay variability. Furthermore, the assay was validated with 80 clinical samples. Real-time PCR showed that 76 out of 80 samples were positive for FAdV-4 (95.0% positivity) while 68 out of 80 were tested positive by conventional PCR (85.0% positivity). Our data suggest this real-time PCR assay could be an attractive tool for screening, confirmatory diagnosis and specific differentiation of FAdV-4 infection.

Vegetative Valvular Endocarditis and Hepatitis Associated with Helcococcus ovis in a 7-year-old White Leghorn Rooster.

Helcococcus ovis is a slow-growing, pyridoxal-dependent, Gram-positive coccus belonging to the Peptostreptococcaceae family. Bacteria belonging to the genus Helcococcus are considered normal inhabitants of keratinized epithelium in humans; however, several reports support their role as pathogens in humans and several animal species. This case report describes the identification of H. ovis in a white leghorn rooster with valvular vegetative endocarditis and hepatitis. In February 2017 one dead, 7-yr-old, white leghorn rooster was submitted to the California Animal Health and Food Safety Turlock laboratory for diagnostic testing. Postmortem and microscopic examination revealed vegetative endocarditis and aortic thrombosis associated with large numbers of Gram-positive cocci. Myocarditis and extensive necrotic hepatitis were also noticed. Helcococcus ovis was isolated in large numbers from the aortic endothelium and confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Bacterial colonies become evident 48 hr postincubation and exhibited a satellite growth around Escherichia coli on blood agar plates. A similar relationship has been described between Helcococcus spp. and Staphylococcus aureus. The primary site of infection in this chicken was not determined. To our understanding this is the first report of H. ovis infection in an avian species. The fastidious nature and nutritional requirements of Helcococcus spp. must be considered in order to allow proper identification and avoid misdiagnosis. Further studies are needed to define pathogenesis, virulence factors, and predisposing conditions associated with this microorganism.

A low cost training phantom model for radio-guided localization techniques in occult breast lesions.

Radio-guided localization (RGL) for identifying occult breast lesions has been widely accepted as an alternative technique to other localization methods, including those using wire guidance. An appropriate phantom model would be an invaluable tool for practitioners interested in learning the technique of RGL prior to clinical application. The aim of this study was to devise an inexpensive and reproducible training phantom model for RGL. We developed a simple RGL phantom model imitating an occult breast lesion from inexpensive supplies including a pimento olive, a green pea and a turkey breast. The phantom was constructed for a total cost of less than $20 and prepared in approximately 10 min. After the first model's construction, we constructed approximately 25 additional models and demonstrated that the model design was easily reproducible. The RGL phantom is a time- and cost-effective model that accurately simulates the RGL technique for non-palpable breast lesions. Future studies are warranted to further validate this model as an effective teaching tool.

Correlation between broiler lameness and anatomical measurements of bone using radiographical projections with assessments of consistency across and within radiographs.

Lameness represents a major welfare and production issue in the poultry industry with a recent survey estimating 27% of birds lame and 3% unable to walk by 40 d of age. A variety of factors may induce lameness and are typically grouped into 2 broad classes on the basis of being infectious or skeletal in nature with the latter accounting for the majority of cases. The current work sought to build upon a large body of literature assessing the anatomical properties of bone in lame birds. Our specific objectives sought to identify relationships between relevant anatomical properties of the tibia and metatarsus using digital quantification from radiographs of legs and a measure of walking difficulty. Resulting output was statistically analyzed to assess 1) observer reliability for consistency in placing the leg during the radiograph procedure and quantification of the various measures within a radiograph, 2) the relationship between the various measurements of anatomical bone properties and sex, bird mass, and gait score, and 3) the relationship between each measurement and leg symmetry. Our anatomical bone measures were found to be reliable (intra-rater and test-retest reliabilities < 0.75) within radiograph for all measures and 8 of the 10 measures across radiographs. Several measures of bone properties in the tibia correlated to difficulty walking as measured by gait score (P < 0.05), indicating greater angulations with increasing lameness. Of the measures that manifested a gait score × bird mass interaction, heavier birds appeared to exhibit less angulation with increasing difficulty walking with lighter birds the opposite. These interactions suggest possibilities for influencing effects of activity or feed intake on bone mineralization with the bone angulation observed. Our efforts agree with that of others and indicate that angulation of the tibia may be related to lameness, though subsequent efforts involving comprehensive measures of bird activity, growth rates, and internal bone structure will be needed if the validity of the measures are to be accepted.

Primary bone tumors in birds: a review and description of two new cases.

Primary bone tumors are only occasionally reported in avian species. This paper presents the cases of an osteosarcoma in a 6-yr-old free-range chicken and a chondrosarcoma in a 3-yr-old barred Plymouth Rock chicken. The well-differentiated, moderately productive osteoblastic osteosarcoma arose from the synsacral vertebrae and had metastasized to the liver. The chondrosarcoma was well differentiated and firmly attached to the left side of the keel. There was no evidence of metastasis.

Biochemical and physiological weaknesses associated with the pathogenesis of femoral bone degeneration in broiler chickens.

Femoral bone degeneration has been recognized as an important cause of lameness in broiler chickens for many years, but the pathogenesis of this condition has not been completely elucidated. The current work presents comprehensive analyses of changes associated with femoral bone degeneration based on findings from gross pathology, histopathology, biochemistry, and synchrotron-based imaging techniques. Gross lesions were predominantly seen in epiphysis and metaphysis of the proximal femur, and infrequently in distal femur, but we did not observe gross lesions in the diaphysis. Bone fractures were observed occasionally, but the most common lesions involved separation of articular cartilage of the femoral bone head, with progressive erosions of the subchondral bone. In advanced cases, on histopathological examination, changes in femoral bone were indicative of chondronecrosis and osteonecrosis. Computed tomography revealed that the degenerative process involves loss of trabecular bone. The course of the lesion development in the mineralized matrix appears to be coupled with increased bone resorption associated with excessive proliferation of pathologically altered osteoclasts. Light microscopy, Fourier transform infrared spectroscopy, and biochemical analysis provided consistent evidence that lowered protein content of the bone organic matrix is an integral component of femoral bone pathology, but these changes do not appear to be associated with excessive activity of matrix metalloproteinases. Taken together, our findings indicate that femoral bone degeneration is associated with structural changes occurring in both inorganic and organic matrix of the bone, but insufficiency in protein metabolism is most probably a primary aetiological factor in the natural history of femoral bone degeneration. However, it is important to stress that our findings do not negate the importance of bacterial infection in the evolution of this condition. Pathogens play a critical role in the progressive pathogenesis of this condition, which ultimately is manifested, in most instances, as femoral head necrosis.

Bone fracture incidence in end-of-lay high-producing, noncommercial laying hens identified using radiographs.

Bone fractures in laying hens are both a welfare and an economic concern for the poultry industry. The aim of this study was to use radiographs to quantify fracture incidence in 6 lines of noncommercial high-producing laying hens. A total of 451 hens (n = 71 to 78) were killed at 47 wk (White Leghorn-Black, White Leghorn-Blue) or 65 wk of age [Barred Plymouth Rock (BR), White Leghorn-Burgundy (WL-BUR), Columbian Plymouth Rock, Rhode Island Red (RIR)]. Radiographs were obtained with hens in 2 positions (lateral and ventrodorsal) and were used to identify fractures in the skeleton. Data on scallop-shaped indentations (possibly fractures) of the keel bone were also collected. After radiography, the left wings were removed for analysis of humeri, radii, and ulnae. Data for the 2 age groups were analyzed separately. The overall incidence of hens with at least 1 fracture was 6.6 and 15.7% in the 47- and 65-wk-old hens, respectively. Fracture incidence in 47-wk-old hens was not different between White Leghorn-Black and White Leghorn-Blue lines. Significant line differences were observed in the 65-wk-old hens, with at least 1 fracture found in 29.5% of RIR hens versus 9.5 and 4.2% observed in Columbian Plymouth Rock and WL-BUR lines, respectively. Fracture incidence in BR hens (18.2%) was greater than in WL-BUR hens. Fractures in RIR hens occurred predominantly in the furculum and wing bones, whereas pubic bones were most affected in BR hens. The proportion of hens with scallop-shaped indentations of the keel ranged from 36.1 to 88.2% and differed between lines in both age groups. High egg production did not seem to be associated with bone fragility in these lines. Two of the older lines (RIR and WL-BUR) had similar egg production, number of eggs to 60 wk, and egg shell weights at 4 ages but had a significantly different fracture incidence. The line differences in fracture incidence may have been affected by calcium metabolism, bone structure, and body weight.

Echocardiographic characteristics of chickens with ascites syndrome.

1. B- and M-mode echocardiography was used to compare cardiac function in broilers with spontaneous ascites syndrome with that of normal chickens. 2. Thirty ascitic chickens and 15 normal chickens aged three, 4, 5, and 6 weeks from the same flock (180 birds in total) were examined. They were restrained gently in a natural standing position, and echocardiographs were obtained from a 7.0-MHz linear transducer placed on the left pectoral apterium. Indices of cardiac structure and functioning were calculated from the echocardiographs, and some were normalised to body weight. Heart rate was also measured. 3. All cardiac structural indices in both ascitic and normal chickens increased with age. Compared with normal chickens, right ventricular diameter at the end of systole in ascitic chickens was greater at 4, 5 and 6 weeks of age. Ventricular septal thickness at the end of both systole and diastole was greater in ascitic chickens at 5 and 6 weeks. Left ventricular free wall thickness at the end of diastole was less in ascitic chickens at 3 weeks. However, all the structural indices decreased with age after normalisation with body weight. 4. The heart rate of ascitic chickens was lower at 4, 5 and 6 weeks. Normalised left ventricular fractional shortening was lower in ascitic chickens at 4, 5 and 6 weeks, as was normalised right ventricular fractional shortening. Incrassation of the ventricular septum (Delta T), which changed little in normal chickens, was less at 4, 5 and 6 weeks in ascitic chickens. Left ventricular fractional shortening, right ventricular fractional shortening and Delta T were all negatively correlated with ascites heart index at all ages. 5. Taken together the results suggest heart failure of both ventricle, but that right ventricular dysfunction is more extensive than left ventricular dysfunction. We suggest that secondary pulmonary hypertension would result in these ascitic chickens due to volume overload.

Pathogenesis of ascites in broilers raised at low altitude: aetiological considerations based on echocardiographic findings.

This study reports novel insight into the aetiology of pulmonary hypertension and ascites in broiler chickens. The scope of measurements was focused on anatomical and functional parameters, and blood flow patterns in leghorns (resistant to ascites), fast-growing broilers (susceptible to ascites), broilers developing ascites, and ascitic broilers evaluated in vivo using echocardiography, and further examined in the context of postmortem findings. Both, in vivo observed features and postmortem findings, showed clear differences between broilers and leghorns, and between normal and ascitic broilers. Abnormalities in the heart chamber geometry and blood flow patterns were detected upon echocardiographic examination in all ascitic broilers. Right and left atrio-ventricular (AV) valve regurgitation were common findings in ascitic broilers and some apparently normal broilers, with left AV valve insufficiency being a predominant feature with respect to degree and frequency of occurrence. Blood flow disturbances were not detected in leghorns. Left ventricular fractional shortening (functional parameter) was considerably reduced (P < 0.01) in ascitic birds (mean: 21.7 +/- 2.0 SE) in comparison with normal broilers (mean: 39.1 +/- 3.6 SE), or leghorns (mean: 43.3 +/- 2.4 SE). The presented findings indicate that pathological and functional changes in the left ventricle and atrium play a significant role in the pathogenesis of ascites in broilers. Severe dilation of the left atrium and pulmonary veins seen on postmortem examination, as well as regurgitant blood flow in the left atrium, demonstrated by Doppler study in ascitic birds, provide evidence that chronically elevated pressure in the left atrium is involved in the aetiology of pulmonary hypertension and ascites in fast-growing broilers.

Activated vitronectin as a target for anticancer therapy with human antibodies.

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites. Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs). In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin. One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/kappa monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues. Iodine 123-radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.