Adenovirus of psittacine birds: investigations on isolation and development of a real-time polymerase chain reaction for specific detection.

Liver samples of psittacine birds with a histological suspicion of an adenovirus infection, confirmed by electron microscopy examination, were subjected to virus isolation attempts using a heterologous cell culture system and a homologous cell culture system in the form of chicken embryo liver cells and psittacine embryo fibroblasts, respectively. Whereas isolation in chicken embryo liver cells failed, virus was isolated successfully in the psittacine embryo fibroblasts cell culture system. Molecular investigations identified the virus as a specific psittacine adenovirus (PsAdV). Additionally, on the basis of the hexon gene sequence data obtained, a real-time polymerase chain reaction (PCR) for specific detection of PsAdV was developed. To ensure an exclusive hybridization with PsAdV, selected primers were located within the variable L1 region of the hexon gene. Furthermore, the specificity of the real-time PCR was confirmed by investigation of a panel of different avian adenoviruses and unrelated DNA viruses. Using this PCR, the threshold cycle values obtained support the propagation of PsAdV in the homologous cell culture system in comparison with the chicken cell culture system. Moreover, the developed PCR represents a reliable method for specific and sensitive detection of PsAdV in clinical samples.

Circovirus-infected geese studied by in situ hybridization.

It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese.

Cloning and sequencing of Duck circovirus (DuCV).

The genome of Duck circovirus (DuCV) is circular and 1996 nts in size. Two major open reading frames were identified, encoding the replicase (V1) and the capsid protein (C1). A stem-loop structure comprising the nonamer 5'-TATTATTAC, conserved in all circo-, nano- and geminiviruses, was found. Unique to DuCV, the region between the 3'-ends of the rep and cap gene contains four repeats of a 44-bp sequence. Phylogenetic analysis shows close relation of DuCV with Goose circovirus and suggests classification of DuCV as a new member of the genus Circovirus of the virus family Circoviridae.

Genome sequence determinations and analyses of novel circoviruses from goose and pigeon.

The genomes of novel circoviruses from goose and pigeon, which were isolated using degenerate primer and inverse primer PCR methods, were cloned and sequenced. Comparative nucleotide (nt) sequence analyses showed that the goose circovirus (GCV) and pigeon circovirus (PiCV) possessed genomes which were 1821 and 2037 or 2036 nt, respectively, and which had features in common with the genomes of porcine circoviruses types 1 and 2 (PCV1, PCV2) and psittacine beak and feather disease virus (BFDV), such that they can now be assigned to the genus Circovirus of the family Circoviridae. Common features include the possession of (i) a potential stem-loop/nonanucleotide motif with which the initiation of rolling circle replication of the virus DNA is associated; (ii) two major ORFs, located on the virus (V1 ORF) and complementary (C1 ORF) strands, which encode the replication-associated protein (Rep) and capsid protein, respectively; (iii) high levels of amino acid identity (41.2--58.2%) shared with other circovirus Rep proteins; and (iv) direct/inverted repeat sequences within the putative intergenic region. On the basis of nt and amino acid sequence identities, GCV is substantially less closely related to BFDV than PiCV is to BFDV.

A diagnostic study on columbid circovirus infection.

A systematic study was performed to examine the frequency of columbid circovirus (CoCV) infection in diseased young pigeons submitted for necropsy and its relevance to pigeon health. Existing diagnostic methods were compared. Among 176 diseased young pigeons examined, CoCV infection was detected in 83 cases using negative contrast electron microscopy. Histopathological examination allowed a clear diagnosis in only 42 pigeons. Therefore, a polymerase chain reaction assay and an in situ hybridization test were developed as additional diagnostic tools. CoCV is by far the most frequently detected infectious agent in diseased young pigeons. Infected pigeons reveal a broad range of concurrent infections. Pathological findings suggest an immunosuppressive effect of CoCV.

Characterisation of PCV-2 isolates from Spain, Germany and France.

The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes the characterisation of PCV-2 isolates from animals affected with PMWS from Germany, Spain and France. We could demonstrate the presence of circovirus by electron microscope, in situ hybridisation and PCR. PCR revealed incidence of PCV-2 in many tissues of one infected animal with the exception of heart and nervous system. The phylogenetic analysis of all PCV-2 isolates yet published in the database, showed relationship of isolates from Spain, Germany and France, with three sequences from Canada determined recently and two isolates from Taiwan, while other North American sequences display a separate cluster. PCR screening of randomly collected organ samples from German pigs not affected with PMWS, revealed a rate of infection with PCV-1 of 5% and with PCV-2 of 26.8%, while blood samples showed a lower incidence.

Cloning and sequencing of columbid circovirus (coCV), a new circovirus from pigeons.

The complete nucleotide sequence of columbid circovirus (CoCV) isolated from pigeons is described. CoCV was amplified using a consensus primer PCR approach directed against conserved sequences within the rep genes of vertebrate circoviruses. The genome of CoCV is circular and 2037 nt in size. It displays 55% homology to the genome of psittacine beak and feather disease virus and is more distantly related (< 40% homology) to porcine circovirus type 1 and 2. Two major open reading frames were identified, encoding the replicase and the putative capsid protein of CoCV. A region similar to the origin of replication of other circoviruses was found: it encompasses a stem-loop structure with the nonamer 5'-TAGTATTAC, conserved in circo-, nano- and geminiviruses. Phylogenetic analyses suggest classification of CoCV as member of the genus Circovirus of the virus family Circoviridae.

A circovirus-like infection in geese related to a runting syndrome.

Diagnostic investigations in a large commercial flock of geese with a history of increased losses and runting, lead to the negative contrast electron microscopic detection of circovirus-like viruses in the lymphoreticular tissue in eight of nine selected geese with growth retardation. With the exception of a mild cloudiness of some air sacs, macroscopic changes pointing to an infection were missed. Histopathological changes concentrated on the lymphoreticular tissue. Lymphocytic depletion and histiocytosis were most evident in the bursa of Fabricius. Basophilic globular inclusions were found in the cytoplasm of medullar and cortical bursal follicular cells, and bursal epithelial cells. Ultrastructural examination of these inclusions revealed paracrystalline or multilayered arrays, or randomly arranged complexes of isometric viral particles, about 14 nm in diameter. The pathological appearence of this, so far unknown, infection in geese shows numerous similarities to circovirus infection in pigeons. In the described flock of geese, a virus-induced immunosuppression could have been a predisposing factor for other infectious agents such as Riemerella anatipestifer or Aspergillus fumigatus, thus contributing to the increased losses and developmental disorders.

Comparison of skeletal muscle characteristics in chicken bred for meat or egg production. I. Histopathological and electron microscopic examination.

A comparative histopathological and electron microscopic study based on selected skeletal muscle in layer-type and meat-type chickens was carried out to detect possible effects of selection for rapid growth and increased muscularity on muscle health. Disseminated fibre degeneration and other myopathological changes occurred more frequently in muscles of meat-type chickens than in layer-type chickens of the same age. The breast region of meat-type chickens--highly developed as a breeding aim--was particularly affected. Experimental exertion demonstrated the limited adaptability of skeletal muscle to psychomotor stress in meat-type chickens. Meat-type chickens showed a higher increase of histopathological muscular lesions after repeated wing exercise than layer-type chickens. Ultrastructural lesions caused by experimental exertion were classified as adaptive-reversible in layer-type chickens. As opposed to this, irreversible destruction of myofibrillar architecture in meat-type chickens are an indication for muscle ischaemia after exertion. Significant changes of structural, metabolic and functional parameters of skeletal muscle related to breeding aims in meat type chickens contribute to finding an explanation for their increased susceptibility to pathological reactions.

Comparison of skeletal muscle characteristics in chicken bred for meat or egg production: II. Histochemical and morphometric examination.

Comparative histochemical and morphometric examinations of layer-type and meat-type chickens were carried out to detect the effects of growth selection on metabolic and structural characteristics of skeletal muscle. A combined histochemical method (NADHOX/mATPase) was used to compare the fibre type profiles of the supracoracoideus muscle in layer-type and meat-type chickens of the same age. A significantly higher percentage of glycolytic fibres was found in the line selected for rapid growth. Very similar results were obtained in studies comparing the supracoracoideus and flexor cruris medialis muscles of layer-type and meat-type chickens with approximately the same body weight. These results indicate that the oxidative capacity of skeletal muscle in meat-type chickens is significantly reduced by a lower proportion of oxidative fibres. The morphometric analysis demonstrates that selective changes of fibre diameters additionally reduce the oxidative capacity. Adverse effects on capillarity and oxygen supply are known to be connected with a reduced oxidative capacity of muscle tissue.

[Adenovirus infections in psittacines]. / Adenovirusinfektion bei Psittaziden.

Diagnostic investigations were carried out in a fatal disease of several African Grey Parrots (Psittacus erithacus) and Cape Parrots (Poicephalus robustus) in a large breeding plant. Electron microscopically examination of liver and intestine, the organs with the most prominent pathomorphological changes, regularly revealed adenoviruses. Necrotizing hepatitis and catarrhal to haemorrhagic enteritis dominated on histopathological examination. Numerous basophilic intranuclear inclusion bodies in hepatocytes and enterocytes and their ultrastructural characteristics underline the etiological role of the detected adenoviruses. Adenoviruses were isolated from livers of three different birds and once from the intestine. Serologically the isolates were classified as fowl adenovirus serotype 4 (FAV4). Restriction enzyme analysis of two isolates showed the identity with the FAV4 reference strain KR5.

[Circovirus infections in pigeons]. / Circovirusinfektion bei Tauben.

The first detection of circovirus infection in pigeons in Germany is described. The existing experiences with this still little known pigeon disease are summarized and similarities to circovirus infections in other species are shown.

Pseudoepidemic of nontuberculous mycobacteria in a community hospital.

Soon after our hospital instituted the BACTEC mycobacterial isolation system, the proportion of cultures positive for nontuberculous mycobacteria increased dramatically. After eliminating other possible sources of contamination, the BACTEC probe's sterilization time and temperature were increased, and the incidence of positive mycobacterial cultures returned to its former rate.

Increased prevalence of Helicobacter pylori antibodies among nurses.

BACKGROUND: Numerous studies have suggested that Helicobacter pylori infection in asymptomatic subjects is transmitted from person to person. Its prevalence is higher in the institutionalized setting. If that is the case, persons involved in patient care should have a higher prevalence of the infection. METHODS: We estimated the prevalence of H pylori antibodies among groups of asymptomatic medical and nursing staff and compared them with volunteer blood donors of similar age and sex. RESULTS: One hundred fifty-eight nurses and aides, 59 residents, 46 senior medical students, and 22 senior nursing students were enrolled in this study. Serum samples were tested for IgG antibodies against H pylori by enzyme-linked immunosorbent assay. Sixty-two (39%) of 158 nurses were found to be positive for antibodies to H pylori compared with 114 (26%) of 441 specimens from the blood donor group. Within the youngest age group (20 to 34 years), 13 (25%) of 51 nurses were positive for H pylori antibodies compared with 19 (13%) of 143 age-matched serum samples from the blood donor group. Within the middle age group (35 to 49 years), 32 (39%) of 83 nurses were positive for H pylori antibodies vs 43 (26%) of 167 age-matched blood donors. In the oldest age group (> 50 years), 17 (71%) of 24 nurses were positive for H pylori antibodies compared with 52 (40%) of 131 age-matched blood donors. Twenty-three (27%) of 86 nurses with 1 to 15 years of occupational exposure were positive for H pylori antibodies compared with 40 (56%) of 72 nurses with more than 15 years of occupational exposure. CONCLUSIONS: Nurses have an increased prevalence of H pylori antibodies that is significantly higher than the comparable prevalence of volunteer blood donors and is evident in the youngest age group. In addition, the increased prevalence is related to a longer duration of patient exposure in the nursing group.