In silico prediction of Gallibacterium anatis pan-immunogens

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0080-0) contains supplementary material, which is available to authorized users.     

The rarely reported tet(31) tetracycline resistance determinant is common in Gallibacterium anatis

The present investigation was undertaken to identify and characterize the tetracycline resistance determinant in 22 Gallibacterium anatis strains for which no determinant was identified using primers specific for tet(A, B, C, D, E, G, H, K, L, M, O). A recent study found tet(B) to be the most prevalent tetracycline resistance determinant in a larger collection of G. anatis field strains from Mexico and Denmark. However, in 41% of the tetracycline resistant strains no determinant could be assigned. Here we demonstrate that tet(31) is a common determinant in G. anatis originating from chickens from very different production systems and localities. In addition, tet(31) was identified in strains isolated over a 30-year period. This is the first report on tet(31) since its original identification in Aeromonas salmonicida.     

Fungal pathogens in Norwegian lettuce fields

Abstract

Norwegian field production of lettuce has increased considerably since the early 1990s. Disease problems rarely required fungicide applications before 1996, when lettuce downy mildew (Bremia lactucae) caused severe losses. From 2002 to 2004, surveys were conducted to identify fungal diseases in Buskerud, Vestfold and Østfold counties in the south-east and Rogaland County in the south-west, representing the main lettuce production regions of Norway. The distribution and incidence of B. lactucae was highly variable, but this pathogen was the most important due to the destructive nature of uncontrolled epidemics. Septoria lactucae caused severe damage, but was found in only one field. Sclerotinia sclerotiorum was the most widespread pathogen, found in 32% of the fields, but usually affecting less than 10% of the plants. Pythium tracheiphilum was reported from 33% of the fields in south-east Norway, but was not found in the south-west. Disease incidence was usually less than 5%, and a disease incidence of more than 10% was reported in one field only. Other pathogens of potential economic importance in Norwegian lettuce fields are Alternaria spp., Botrytis cinerea and Rhizoctonia solani, although they were sporadically distributed in relatively few fields in this survey.     

Effect of Cultivar Resistance and Haulm Killing Method on Tuber Infection by <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

A series of field trials combining cultivars with different levels of resistance to Phythophtora infestans and different haulm killing methods was conducted in 1997, 1998 and 1999 to assess the contamination potential of the haulm. In addition, the frequency of tubers with late blight was assessed after harvest and storage, combining effects of pretreatment infection, infection by the haulm at harvest and the development of the disease during storage. Haulm killing was performed by diquat (200, 400, 600 g a.i. ha^-1), half cutting of the haulm in combination with diquat (100, 200, 300 g a.i. ha^-1) or full cutting of the haulm. In those treatments where haulm killing was performed purely chemically the haulm remnants’ contamination potential tended to increase at reduced doses. Full mechanical destruction gave as low a contamination potential as application of the full dose of chemical haulm killer. However, there were no consistent differences between the haulm killing treatments in the frequency of blighted tubers after harvest and storage. This indicates that the input of chemical haulm killers may be reduced. There was a large difference between years in tuber blight after harvest and storage, which confirms that climatic factors are of major importance for tuber infection. The large difference found between the cultivars in the frequency of tuber blight indicates that tuber resistance should be an important part of integrated late blight disease management.     

Identification of Actinobacillus Pleuropneumoniae Serotype 2 by Monoclonal or Polyclonal Antibodies in Latex Agglutination Tests

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a major problem in modern swine industry worldwide. At present 12 serotypes have been recognized (Nielsen 1990). Serotyping has been based upon capsule associated, heat-stabile antigens of polysaccharide (PS) and lipopolysaccharide (LPS) nature. A variety of tests have been used for serotyping including slide agglutination (Mittal et al. 1982), immunodiffusion (Gunnarsson et al. 1978, Nielsen & O’Connor 1984), indirect haemagglutination (Mittal et al. 1983a) and coagglutination (Mittal et al. 1983b). Recent studies report on serotyping of A. pleuropneumoniae strains using monoclonal antibodies in enzyme-linked immunosorbent assays (Lida et al. 1990, Nakai et al. 1990).     

Pigmentation and spectral absorbance signatures in deep-water corals from the Trondheimsfjord, Norway

The pigmentation and corresponding in vivo and in vitro absorption characteristics in three different deep-water coral species: white and orange Lophelia pertusa, Paragorgia arborea and Primnoa resedaeformis, collected from the Trondheimsfjord are described. Pigments were isolated and characterized by High-Performance Liquid Chromatography (HPLC) analysis and High-Performance Liquid Chromatography Time-Of-Flight Mass Spectrometer (LC-TOF MS). The main carotenoids identified for all three coral species were astaxanthin and a canthaxanthin-like carotenoid. Soft tissue and skeleton of orange L. pertusa contained 2 times more astaxanthin g(-1) wet weight compared to white L. pertusa. White and orange L. pertusa were characterized with in vivo absorbance peaks at 409 and 473 nm, respectively. In vivo absorbance maxima for P. arborea and P. resedaeformis was typically at 475 nm. The shapes of the absorbance spectra (400-700 nm) were species-specific, indicated by in vivo, in vitro and the corresponding difference spectra. The results may provide important chemotaxonomic information for pigment when bonded to their proteins in vivo, bio-prospecting, and for in situ identification, mapping and monitoring of corals.     

Podzol development with time in sandy beach deposits in southern Norway

The coastal areas of SE Norway provide suitable conditions for studying soil development with time, because unweathered land surfaces have continuously been raised above sea level by glacio‐isostatic uplift since the termination of the last ice age. We investigated Podzol development in a chronosequence of six soils on sandy beach deposits with ages ranging from 2,300 to 9,650 y at the W coast of the Oslofjord. The climate in this area is rather mild with a mean annual temperature of 6°C and an annual precipitation of 975 mm (Sandefjord). The youngest soil showed no evidence of podzolization, while slight lightening of the A horizon of the second soil (3,800 years) indicated initial leaching of organic matter (OM). In the 4,300 y–old soil also Fe and humus accumulation in the B horizon were perceptible, but only the 6,600 y–old and older soils exhibited spodic horizons. Accumulation of OM in the A horizons reached a steady state in <2,300 y, while in the B horizons OM accumulated at increasing rates. pH dropped from 6.6 (H₂O)/5.9 (KCl) in the recent beach sand to 4.5 (H₂O)/3.8 (KCl) within approx. 4,500 y (pH_H2O)/2,500 y (pH_KCl) and stayed constant thereafter, which was attributed to sesquioxide buffering. Base saturation showed an exponential decrease with time. Progressive weathering was reflected by increasing Fe_d and Al_d contents, and proceeding podzolization by increasing amounts of pyrophophate‐ and oxalate‐soluble Fe and Al with soil age. These increases could be best described for most Fe and Al fractions by exponential models. Only the increasing amounts of Fe_p could be better described by a power function and those of Fe_o by a linear model.     

Antimicrobial susceptibility and tetracycline resistance determinant genotyping of Gallibacterium anatis

The present investigation was undertaken to assess the antimicrobial susceptibility of a collection of 58 Gallibacterium isolates. All strains were tested by the broth dilution method using the veterinary fastidious medium. A total of 46 field strains were tested, whereof 23 were clinical isolates from 10 Mexican layer flocks and another 23 isolates originated from 13 clinically healthy poultry flocks in Denmark. In addition, 12 Gallibacterium reference strains that had been isolated some 30–40 years ago were included.