Teaching the "hybrid approach": a novel swine model of muscular ventricular septal defect.

This report describes a reproducible swine model for creating muscular ventricular septal defects (VSDs). The model not only facilitates the development and modification of hybrid techniques for closing muscular VSDs, but also serves as a teaching tool that allows operators to become accustomed to the specific technical requirements necessary when using the hybrid approach to perform periventricular VSD device closure. The authors' institutional experience using this novel animal model is presented.

Growth analysis of adenoviruses isolated from pigeons in chicken cells and serological characterization of the isolates.

The susceptibility of chicken embryo liver (CEL) and chicken kidney (CK) cells to eight adenoviruses isolated from different pigeons were investigated. Isolation and propagation was most successful in CEL cells. The cytopathic effect, i.e. cell rounding and detachment of the cells was typical for adenoviruses. Titres of up to 10(6.6) plaque-forming units/ml could be reached on CEL cells. Transferring CEL-grown virus onto CK cells also resulted in a cytopathic effect but with much lower titres, whereas the isolation of pigeon adenoviruses on these cells was not successful. Antibodies against fowl adenovirus reference strains (FAV1-12) were used to serotype the isolates in neutralisation tests. Six were identified as FAV serotypes 2,5,6,10 and 12. Two isolates could not be typed. An antiserum produced in chickens against one of these untypable strains was able to neutralize both. The neutralization indexes of these strains were very similar, indicating that they are probably the same serotype. A cross neutralization test confirmed that this serotype is different from known fowl adenoviruses.

Identification and characterization of penton base and pVIII protein of egg drop syndrome virus.

The nucleotide sequence and location of the penton base and pVIII genes of the egg drop syndrome (EDS) virus an avian adenovirus, were determined. The penton base gene is located at 34.8-38.8 map units. The coding sequence has a length of 1359 nt and encodes a polypeptide of 452 amino acids (aa) with a molecular weight of 51 105 Da. The amino acid sequence shows a homology of 57.3 and 55.3% to the structural protein IH of human adenovirus serotype 2 (HAd2) and fowl adenovirus serotype 1 (FAV1). The penton base protein lacks the integrin binding motifs RGD (Arg-Gly-Asp) and LDV (Leu-Asp-Val). At 65.1-67.3 map units an open reading frame of 753 nucleotides was identified which encodes the structural protein pVIH. It forms a protein of 250 aa with an expected molecular weight of 28 501 Da. Possible protease cleavage sites were identified. Amino acid homologies of 30.8 and 27.7% were found to the HAd2 and FAV1 pVIII genes. Remarkably, the overall amino acid identity with ovine adenovirus pVIII is 52%. The start codons of both genes are shifted leftwards compared with the respective structural elements on the HAd2 or FAV1 genomes which indicates a different genomic organization of the EDS virus.

Identification and characterization of an avian adenovirus isolated from a 'spiking mortality syndrome' field outbreak in broilers on the Delmarva Peninsula, USA.

A fowl adenovirus isolated from a 'spiking mortality syndrome' field outbreak in broilers on the Delmarva Peninsula, USA, was identified by cross-neutralization tests and by restriction enzyme analysis using eight restriction enzymes as a strain of the fowl adenovirus serotype FAV12. The isolate is nearly identical to strain UF71 though it shows substantially higher virulence in vivo than UF71. Contrary to strains of other fowl adenovirus serotypes (except serotype FAV1), the isolate caused high embryo mortality in specific pathogen free (SPF) chicken embryos inoculated via the allantoic cavity. Infection of 83 one-day-old SPF chicks with 105 plaque forming units of the isolate via a natural route led to clinical signs and growth inhibition. Gross and histological lesions in the liver, bursa of Fabricius and thymus, and in five cases hydropericarditis were observed. An inclusion body hepatitis was found in two chicks.

Molecular characterization of two strains of the avian adeno-associated virus (AAAV).

An avian adeno-associated virus (AAAV) was isolated after propagating a field isolate of the CELO virus (fowl adenovirus serotype 1 (FAV1)) in embryonated eggs. The isolated dependovirus was compared with the known AAAV obtained from the American Type Culture Collection (ATCC VR-865). The genomes were analysed by digestion with several restriction endonucleases. Although both DNAs have the same size, most restriction enzymes produced different restriction patterns. Double digests were used to construct for the first time restriction maps for avian dependoviruses. The two DNAs rendered different restriction maps in which the different restriction sites were mainly located in the middle and right part of the genomes. The effect of these differences on the structure proteins was shown by western blot analysis. In the immunoblot, the immunofluorescence and immunodiffusion test the two dependoviruses were serologically indistinguishable and therefore can be regarded as two different strains of the same virus. To differentiate between both strains we named the original one as AAAV VR-865 compared with the isolated AAAV DA-1.

[Pathogenicity of avian adenoviruses in chickens]. / Zur Frage der Pathogenität der aviären Adenoviren beim Huhn.

In the last years avian adenoviruses have caused diseases with similar symptoms and high losses in four countries. The causative viruses could be grouped by cross-neutralization tests and restriction enzyme analysis into four serotypes. Within the serotypes there exist strains with pathogenic properties as well as strains without pathogenicity. Epidemiologic investigations demonstrated that not only immunosuppression or co-infections produce diseases after adenovirus infections. Vertical transmission of the virus from parent stocks and deficiency of specific maternal antibodies in the chickens contribute to the development of diseases. Parent stocks, which were infected with different serotypes of adenovirus during their rearing period, produce chickens with maternal antibodies. These chickens are protected against early infections with avian adenoviruses.

Identification of genomic regions of the herpesvirus of turkeys (HVT) with helper activity for avian adeno-associated virus (AAAV).

Herpesvirus of turkeys (HVT) is a potent helper for the defective parvovirus avian adeno-associated virus (AAAV). To study the helper mechanism at the molecular level, we established a complete cosmid library of HVT DNA in a set of seven overlapping clones and transiently cotransfected secondary chicken embryo fibroblast (CEF) cells with AAAV DNA and recombinant cosmids (cBL) (individual as well as in different combinations). Using an AAAV-specific indirect immunofluorescence assay, we identified four regions on the HVT genome, represented by cBL267, cBL27, cBL33, and cBL34, which express helper functions for AAAV. As demonstrated by infection studies with extracts from cotransfected CEF cells, cBL267 promotes productive AAAV growth, while the helper effect induced by cBL27, cBL33, and cBL34 is limited to the synthesis of noninfectious AAAV antigen. In view of the data presented, possible HVT-specific helper mechanisms for AAAV are discussed.

[The distribution of avian enteroviruses in Germany]. / Untersuchungen zur Verbreitung aviärer Enteroviren in Deutschland.

Sera from chicken flocks of different regions in Germany were examined for antibodies to avian nephritis virus (ANV) by indirect immunofluorescence. There was no evidence of a wide distribution of ANV, at least in chicken parent flocks, in contrast to results from investigations in England, Northern-Ireland and Japan.

Biological and physicochemical characterization of the major (1.40) and minor (1.45) component of infectious avian adeno-associated virus.

Two infectious components with buoyant densities of 1.40 g/cm3 and 1.45 g/cm3, designated as major (1.40) and minor (1.45) component, were detected by banding avian adeno-associated virus (AAAV) isopycnically in CsCl. In metrizamide, however, infectious AAAV banded only as a single peak at a density of 1.32 g/cm3. Biological as well as physicochemical properties of the two AAAV components recovered from CsCl density gradient were described. Concerning the minor (1.45) component, three experimental findings may suggest that the capsid structure of this AAAV population is altered in comparison with that of the major (1.40) component: (i) the sedimentation pattern characterized by an additional peak containing slower-sedimenting noninfectious material (16 S); (ii) the specific infectivity decreased by the 3.5 fold; (iii) the ready disintegration when exposed to gently denaturing conditions.

Growth of avian adeno-associated virus in chicken cells transfected with fowl adenovirus serotype 1 DNA.

Using the chloroquine-modified calcium phosphate coprecipitation technique, fowl adenovirus serotype 1 (FAV 1) DNA transfects efficiently chicken cell cultures. Infection of FAV 1 DNA transfected cells with helper dependent avian adeno-associated virus (AAAV) results in the production of AAAV progeny, being detected by an indirect immunofluorescence assay. These findings indicate that FAV 1 DNA introduced into the host cell promotes actively the growth of AAAV.

[Infectious disease factors in poultry]. / Infektiöse Faktorenkrankheiten beim Geflügel.

The increased importance of multicausal diseases of poultry in the present time is due to a better control and prevention of the monocausal diseases and epidemics predominant in the past. This development is supported by the intensive methods of modern poultry husbandry, the high stocking densities and the improved performance of the poultry by genetic selection. Opportunistic as well as pathogenic organisms can be involved in the development of infectious factorial diseases. A variety of clinical signs can be observed. Above all immunosuppression accompanied by mixed infections and other factors causes health problems of great economical importance. Frequently series of events take place in the development of infectious factorial diseases, in the course of which a preceding disturbance acts as factor of further diseases. Some examples of diseases and syndroms are given to substantiate the multiple involvement of different factors and infections in the development of series of events.

Avian adeno-associated parvovirus and Marek's disease virus: studies of viral interactions in chicken embryo fibroblasts. Brief report.

Using growth kinetics we demonstrate two effects based on interactions between the chicken herpesvirus, Marek's disease virus (MDV), and the dependovirus, avian adeno-associated virus (AAAV), in coinfected chicken embryo fibroblasts (CEF): (i) MDV provides helper activity for an efficient multiplication of AAAV; (ii) a high multiplicity of coinfecting AAAV inhibits completely the growth of MDV as well as AAAV.

Herpesviruses provide helper functions for avian adeno-associated parvovirus.

The avian herpesviruses infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as the mammalian herpesvirus pseudorabies virus (PRV) were able to provide complete helper activity for the production of infectious avian adeno-associated virus (AAAV) in chicken cells. The presence of AAAV in the infected chicken cell reduced the multiplication of HVT. ILTV or PRV, however, were not affected if used as helper viruses. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious herpesvirus by plaque assays.

Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): studies of viral interactions in chicken cell cultures.

As the growth kinetics of avian adeno-associated virus (AAAV) in chicken cells demonstrate, the three serotypes of fowl adenovirus (FAV), FAV -1, -5 and -8, provide complete helper activity for the production of infectious AAAV. Under one step conditions, the growth cycle of AAAV in primary chicken kidney cell (CKC) cultures is characterised by an eclipse phase of 8 hours and an exponential increase of the virus infectivity by 4 to 5 logs until 24 hours post-adsorption (p.a.). These growth characteristics do not depend on the serotype of FAV used as helper. In chicken embryo fibroblast (CEF) cultures the eclipse phase is prolonged to 12 hours p.a. and the virus infectivity increases only by 2 logs. In addition, the low efficiency of plating of FAV -1 in this cell system does not allow one step growth curves for AAAV. In CKC and CEF cultures coinfected with FAV and AAAV the multiplication of helper FAV is reduced. The degree of growth inhibition depends on the AAV multiplicity used. Sequential infection of CKC cultures with FAV -1 and AAAV modifies the AAAV growth cycle, i.e. there is a time reduction of the eclipse phase and a decrease of the virus yield. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious FAV by a plaque assay.

Comparison of the enzyme-linked immunosorbent assay (ELISA), haemagglutination inhibition test and agar gel precipitation test for detection of antibodies to avian infectious bronchitis virus.

The immune response after vaccination with infectious bronchitis virus (IBV) under field conditions was measured by the ELISA, haemagglutination-inhibition (HI) and agar-gel precipitin (AGP), tests. Vaccinations were performed in three flocks and one experimental group via the drinking water with the vaccine strains H 120 and H 52. In each flock 40 random serum samples were taken every 2 weeks and tested individually. In the experimental group blood samples were collected every week from each of the 10 chickens. The primary vaccination with H 120 resulted in a rapid increase of antibody titre as detected by ELISA followed by a slow decrease over the next few weeks. By the HI and AGP tests no antibody responses could be seen after this primary vaccination. Revaccination with the H 52 strain provoked a further increase in ELISA titres. In the experimental group, and in flock W, a similar increase occurred by the HI test and precipitating antibodies appeared. The formation of HI antibodies in flock T (nipple waterers) was somewhat retarded and precipitating antibodies were just detectable. In flock F revaccination did not result in the immediate production of HI and AGP antibodies. However, 6 weeks after revaccination a significant rise in ELISA, HI and AGP antibodies was observed, probably as the result of a field infection. It was demonstrated that, based on the higher sensitivity, the ELISA test is more suitable than HI and AGP to monitor antibody responses to vaccination against infectious bronchitis. Strain specificity in the HI test is discussed as a reason for its failure to detect antibodies after primary vaccination with the highly attenuated vaccine strain H 120.

Purification method for the avian adeno-associated virus.

Avian adeno-associated virus (AAAV) pre-purified by Uvasol extraction, one discontinuous and two CsCl equilibrium density gradients, was still considerably contaminated with avian adenovirus (CELO strain). Four different approaches were investigated in attempts to improve the elimination of the contaminating CELO virus. The contaminations were assayed by double immunodiffusion and indirect immunofluorescence. The immunoprecipitation of CELO virus with antiserum and protein A-Sepharose is the most effective method of obtaining purified AAAV free of CELO virus.