Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides (cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.     

Mycobacterium avium subsp. paratuberculosis enters the small intestinal mucosa of goat kids in areas with and without Peyer's patches as demonstrated with the everted sleeve method

The main lesions of paratuberculosis in ruminants are in the small intestine. Previous studies have shown that the bacterium enters the small intestine through M cells found in the follicle-associated epithelium lining the domes of the Peyer's patches. The everted sleeve method, devised for the in vitro study of intestinal absorption, was used in this study to investigate the uptake of Mycobacterium avium subsp. paratuberculosis in goat intestine. Everted small intestinal sleeves of goat kids, prepared from areas with and without Peyer's patches, were incubated for 60 min in 3H-labeled bacterial solution. The results of this study imply that the bacteria can enter the intestinal mucosa of the jejunum, both in areas with and without Peyer's patches. These findings indicate, therefore, that M. avium subsp. paratuberculosis bacteria not only enter through M cells but also through enterocytes.     

Drag force acting on biofouled net panels

Measurements were made to assess the increase in drag on aquaculture cage netting due to biofouling. Drag force was obtained by towing net panels, perpendicular to the incident flow, in experiments conducted in a tow tank and in the field. The net panels were fabricated from netting stretched within a 1 m² pipe frame. They were towed at various speeds, and drag force was measured using a bridle-pulley arrangement terminating in a load cell. The frame without netting was also drag tested so that net-only results could be obtained by subtracting out the frame contribution. Measurements of drag force and velocity were processed to yield drag coefficients.

Clean nets were drag tested in the University of New Hampshire (UNH) 36.5 m long tow tank. Nets were then exposed to biofouling during the summer of 2004 at the UNH open ocean aquaculture demonstration site 1.6 km south of the Isles of Shoals, New Hampshire, U.S.A. Nine net panels were recovered on 6 October 2004 and immediately drag tested at sea to minimize disturbing the fouling communities. The majority of the growth was skeleton shrimp (Caprella sp.) with some colonial hydroids (Tubularia sp.), blue mussels (Mytilus edulus) and rock borer clams (Hiatella actica). Since the deployment depth was 15 m (commensurate with submerged cages at the site), no algae were present. The net panels had been subject to several different antifouling treatments, so the extent of growth varied amongst the panels. Drag force measurements were made using a bridle-pulley-load cell configuration similar to that employed in the tow tank. Fixtures and instruments were mounted on an unpowered catamaran that was towed alongside a workboat. Thus, the catamaran served as the “carriage” for field measurements.

Increases in net-only drag coefficient varied from 6 to 240% of the clean net values. The maximum biofouled net drag coefficient was 0.599 based on net outline area. Biofouled drag coefficients generally increased with solidity (projected area of blockage divided by outline area) and volume of growth. There was, however, considerable scatter attributed in part to different mixes of species present.     

Immunohistochemical detection of piscine reovirus (PRV) in hearts of Atlantic salmon coincide with the course of heart and skeletal muscle inflammation (HSMI)

ABSTRACT: Aquaculture is the fastest growing food production sector in the world. However, the increased production has been accompanied by the emergence of infectious diseases. Heart and skeletal muscle inflammation (HSMI) is one example of an emerging disease in farmed Atlantic salmon (Salmo salar L). Since the first recognition as a disease entity in 1999 it has become a widespread and economically important disease in Norway. The disease was recently found to be associated with infection with a novel reovirus, piscine reovirus (PRV). The load of PRV, examined by RT-qPCR, correlated with severity of HSMI in naturally and experimentally infected salmon. The disease is characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of the red skeletal muscle. The aim of this study was to investigate the presence of PRV antigens in heart tissue of Atlantic salmon and monitor the virus distribution in the heart during the disease development. This included target cell specificity, viral load and tissue location during an HSMI outbreak. Rabbit polyclonal antisera were raised against putative PRV capsid proteins μ1C and σ1 and used in immunohistochemical analysis of archived salmon heart tissue from an experimental infection. The results are consistent with the histopathological changes of HSMI and showed a sequential staining pattern with PRV antigens initially present in leukocyte-like cells and subsequently in cardiomyocytes in the heart ventricle. Our results confirm the association between PRV and HSMI, and strengthen the hypothesis of PRV being the causative agent of HSMI. Immunohistochemical detection of PRV antigens will be beneficial for the understanding of the pathogenesis of HSMI as well as for diagnostic purposes.     

Development of an improved species specific PCR test for detection of Haemophilus parasuis

A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold lower when tested on pure cultures of H. parasuis (5CFU and 0.5CFU/PCR reaction, respectively). Addition of 1.4 x 10(5) Escherichia coli to each PCR tube did not alter the sensitivity of the tests. No difference in sensitivity of the tests was observed when tested on purified DNA. On the other hand, the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test of Oliveira et al. is used on samples from systemic locations the chances for false positive results are apparently low. The present PCR test represents a rapid and reliable method for genetically based identification of H. parasuis. The high species specificity of the test makes it suitable for detection of H. parasuis in clinical samples, regardless of the presence of affiliated species and contaminating flora. As the two PCR tests differ in sensitivity and specificity, the use of both PCR tests for different purposes is a possibility.     

Climatic control of bud burst in young seedlings of nine provenances of Norway spruce

Detailed knowledge of temperature effects on the timing of dormancy development and bud burst will help evaluate the impacts of climate change on forest trees. We tested the effects of temperature applied during short-day treatment, duration of short-day treatment, duration of chilling and light regime applied during forcing on the timing of bud burst in 1- and 2-year-old seedlings of nine provenances of Norway spruce (Picea abies (L.) Karst.). High temperature during dormancy induction, little or no chilling and low temperature during forcing all delayed dormancy release but did not prevent bud burst or growth onset provided the seedlings were forced under long-day conditions. Without chilling, bud burst occurred in about 20% of seedlings kept in short days at 12 degrees C, indicating that young Norway spruce seedlings do not exhibit true bud dormancy. Chilling hastened bud burst and removed the long photoperiod requirement, but the effect of high temperature applied during dormancy induction was observed even after prolonged chilling. Extension of the short-day treatment from 4 to 8 or 12 weeks hastened bud burst. The effect of treatments applied during dormancy development was larger than that of provenance; in some cases no provenance effect was detected, but in 1-year-old seedlings, time to bud burst decreased linearly with increasing latitude of origin. Differences among provenances were complicated by different responses of some origins to light conditions under long-day forcing. In conclusion, timing of bud burst in Norway spruce seedlings is significantly affected by temperature during bud set, and these effects are modified by chilling and environmental conditions during forcing.     

Serological characterization of Danish Haemophilus parasuis isolates

A total of 103 Danish Haemophilus parasuis field isolates was collected from diseased pigs in connection with routine diagnostics. The isolates were serotyped using indirect haemagglutination (IHA) and for 57 of the isolates the serotyping was also performed by immunodiffusion. Serovar 5 was the most prevalent (36%), followed by serovar 4 (13%) and serovar 13 (22%), whereas 15% of the strains were nontypeable by IHA. Serovars 1, 2, 6, 7, 9, 12, 14, and 15 were only represented by a small number of isolates. Most of the Danish isolates belong to serovars, which earlier have been shown to be virulent. The strains could be divided into two groups depending on whether they were isolated from cases with systemic disease (polyserositis, arthritis or meningitis) or if they only were found in the lower respiratory tract. The most marked differences were observed for serovar 4, which had a higher prevalence in respiratory disease compared to systemic infection, and for the nontypeable isolates, which were mainly found in cases of systemic infection.