Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.     

Ultrasonographic and histologic evaluation of medial and middle patellar ligaments in exercised horses following injection with ethanolamine oleate and 2% iodine in almond oil

OBJECTIVE: To describe the ultrasonographic and quantitative histologic effect of injecting 2% iodine in almond oil (IAO) and ethanolamine oleate (EO) in the medial and middle patellar ligaments of horses and to determine whether a difference in response exists between IAO and EO treatment. ANIMALS: 10 healthy horses. PROCEDURE: In 5 horses, the medial and middle patellar ligaments of 1 limb were injected with EO, whereas IAO was injected in the medial and middle patellar ligaments of another 5 horses. Ultrasonographic evaluation was performed on the experimental and control limb before injection of IAO and EO and prior to euthanasia to determine cross-sectional area and evaluate fiber pattern. The patellar ligaments were harvested 2 weeks after injection and examined histologically to evaluate the inflammatory response, fibroplasia, and chondroid metaplasia. RESULTS: Injection of the patellar ligaments with IAO resulted in a greater increase in cross-sectional area on ultrasonography than EO. Both agents caused a decrease in echogenicity of the ligament. Histologically, significantly greater infiltration of inflammatory cells and fibroplasia developed after injection with IAO, compared with EO. Both agents resulted in significantly greater fibroplasia relative to control specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Injection of the medial and middle patellar ligaments with IAO induces more severe inflammation and fibroplasia than EO. Maturation of the inflammatory and fibrous response may contribute to resolution or attenuation of upward fixation of the patella by subsequent stiffening of the ligaments.     

Concurrent West Nile virus and mycobacterium avium infection in a black-necked swan (Cygnus melanocoryphus).

An adult male black-necked swan (Cygnus melanocoryphus) was evaluated for a routine physical examination and West Nile virus vaccination. Nine days later, the swan was hospitalized for weakness, dehydration, tremors, and leukocytosis. Gradual clinical improvement was seen over a 2-wk course of supportive care. However, the animal became acutely weak and was found dead. Histopathology revealed severe granulomatous pneumonia and air sacculitis with dissemination to the liver, spleen, intestine, and coelom. Mycobacterial infection was confirmed by identification of acid-fast bacilli within tissue sections and by polymerase chain reaction. Concurrent West Nile virus infection was identified in sections of brain by using immunohistochemistry.     

Land-cover and structural changes in a western Norwegian cultural landscape since 1865, based on an old cadastral map and a field survey

Many studies of land-cover and structural changes in cultural landscapes have used historical maps as a source for information about past land-cover. All transformations of historical maps onto modern coordinate systems are however burdened with difficulties when it comes to accuracy. We show that a detailed land survey of the present landscape may enable transformation of an old cadastral map directly onto the present terrain with very high accuracy. The detailed resulting map enabled us to locate remnants of semi-natural grasslands and man-made structures with continuity from 1865 and to test hypotheses about relationships between landscape changes and landscape characteristics. The main land-cover change 1865–2002 was decrease of arable fields, and addition of three new land-cover classes: horticultural, orchard and abandoned areas. Of the 330 man-made structures present in 1865, only 58 remained in 2002, while 63 new structures had been built after 1865. We found that semi-natural grasslands with continuity since 1865 were situated on ground with significantly lower production capacity than mean 1865 production capacity. The man-made structures with continuity since 1865 were also associated with areas with significantly lower production capacity than the 1865 mean, situated in significantly steeper terrain but not further from the hamlet. Our study illustrates the potential of digitised and accurately transformed historical cadastral maps combined with detailed field surveys for analysis of land-cover and structural changes in the cultural landscape.     

Selection experiments with salmon: IV. Growth of Atlantic salmon during two years in the sea

Brood stock of Atlantic salmon were sampled from 37 rivers or riversystems (strains) for three consecutive years. Each strain was represented by several full- and half-sib families. During the freshwater period each full-sib family was reared separately and each group was given as equal environmental conditions as possible. Fingerlings large enough to become 1-year-old smolt were freeze-branded and smolts from the different strains were sampled and transported to five salmon farms along the Norwegian coast. This paper deals with body weight and length of Atlantic salmon after a growth period of 2 years in the sea.Significant differences between strains were found for all three year classes. The interaction farm × strain was significant but accounted for only a small part of the total variation. It is concluded that genotype × environment interaction can be ignored when planning a selection programme for Altantic salmon for Norwegian farming conditions. A significant interaction sire × dam was found for the 1972 year-class, implying the presence of non-additive genetic variation. The heritabilities for body weight and length estimated from sire components (year-class 1973 and 1974) were quite high: 0.31 and 0.28, respectively. Genetic correlations between body weight and length were close to unity. The heritabilities for condition factor ($\text{K=}\text{weight in g}\text{(length in cm)}{}^3\text{·100}$ were small and none of the sire components were significant. It is concluded that the combination of weight and length used in calculating the K-factor has minor genetic basis.Genetic variation in body weight of Atlantic salmon is high, and about three times as high as for body length.     

Mites of the genus Carabodes (Acari,Oribatida) in Norwegian coniferous forests: occurrence in different soils,vegetation types and polypore hosts

Oribatid mites (Acari) represent a considerable part of the biodiversity in Fennoscandian forests, but our knowledge about their habitat requirements is limited. We studied 10 Carabodes species in the forest floor of seven coniferous forest types, and in dead fruiting bodies (sporocarps) of 6 species of wood-living polypore fungi in southern Norway. The most common Carabodes species in soil were rare in sporocarps, and vice versa. The density of several ground-living species was significantly influenced by vegetation type and soil type. Carabodes willmanni and C. subarcticus were considered as lichen feeders on the ground, and occurred abundantly in Cladonia-rich pine forests. Three species, C. femoralis, C. areolatus and C. reticulatus, seem to be sporocarp specialists. Their relative numbers were rather similar in dead sporocarps of five different fungal species, including annual and perennial sporocarps, soft and hard. This was in contrast to beetles from the same sporocarps, which in a previous study proved to be strongly host-specific. Although being tolerant to different fungal species, the association of certain Carabodes species to dead sporocarps could make them vulnerable in forests with little dead wood and few sporocarps.     

Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus morhua L.): The effect of fish meal, standard soybean meal and a bioprocessed soybean meal

Populations of aerobic heterotrophic bacteria present in the gastrointestinal (GI) tract of healthy Atlantic cod (Gadus morhua L.) fed three different diets, fish meal, standard or a bioprocessed soybean meal (BPSBM), were estimated using the dilution plate technique. A total of 944 isolates were characterised by biochemical and physiological properties and 425 isolates were identified further by sequencing the 16S rRNA genes. Our results showed that gut microbiota were affected by dietary manipulation. The GI tract of fish fed fish meal was dominated by Gram-positive bacteria of the genera Brochothrix and Carnobacterium. The Gram-negative bacteria Chryseobacterium spp. and Psychrobacter glacincola, and Gram-positive bacteria belonging to Carnobacterium, dominated in the digestive tract of fish fed soybean meal. In contrast to these results, genus Psychrobacter dominated in the GI tract when fish were fed BPSBM.Until recently, it was generally suggested that the gut microbiota of fish were less diverse than in homoeothermic animals. However, the present study identified several “new” bacterial species isolated from the alimentary tract of Atlantic cod. These “new” bacterial species are not normally isolated from the GI tract of fish. Based on our finding we suggest that the GI tract microbiota of fish might not be as simple as believed.Antagonistic activity of carnobacteria regarding inhibition of growth of two fish pathogens (Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum) was observed. However, some difference in the antibacterial activity of Carnobacterium spp. was observed. Whether this antagonistic activity has any effect in challenge studies will be discussed, especially in relation to the finding that the digestive tract is one of the major infection routes in fish.     

Nitrogen isotope analysis of free glycine in soil using isotope dilution mass spectrometry

To enable the estimation of production and consumption rates of free glycine in soils through ¹⁵N isotope dilution experiments, an isotope dilution mass spectrometric method was developed. The method, which enabled high precision N isotope ratio determination of glycine in soil extracts at δ¹⁵N levels up to 4000‰ and concentrations from approximately 2 μM, is based on the following steps: (i) addition of glycine spike to the soil extract, (ii) removal of humic substances and pre-concentration of glycine using solid phase extraction, (iii) derivatization of amino acids, (iv) separation of the derivatives using gas chromatography (GC), (v) their combustion to yield sample N₂ gas, and (vi) finally the use of N isotope ratio mass spectrometry (IRMS). Judging by uncertainty budget calculations, the precision obtained (SD=0.01-0.06 at% ¹⁵N) is sufficient for detecting differences in N isotopic ratios obtained in ¹⁵N isotope dilution experiments.     

Phosphatase activity, microbial phosphorus, and fine root growth in forest soils in the Sierra de Gata, western central Spain

Acid phosphatase activity (APA), labile P fractions and fine root growth were studied in an oak (Quercus pyrenaica Willd.) forest in the Sierra de Gata, in western central Spain. Soils in the region are acid and rich in organic matter, with low levels of extractable inorganic P but with a high proportion of organic P. In such soils, the activity of phosphatase enzymes is likely to be important for the control of P mineralization and P cycling and, consequently, can affect the availability of P for plant uptake. The biomass of fine roots was about 25-fold that of leaf litter, demonstrating a high allocation of C resources to the root system in order to compensate for a low availability of soil nutrients. The study compared plots fertilized with triple superphosphate (100 kg P ha^-1) to control (unfertilized) plots. Fertilizer application had no significant effect on APA and fine root density; however, there were significant differences in available and microbial P. Spatial and seasonal variations in the APA were related to plant root density and biotic demand. Seasonal differences in the APA may also be the consequence of changes in the amount of hydrolysable organic substrates at different times of the year.     

Surgical amputation of a digit and vacuum-assisted-closure (V.A.C.) management in a case of osteomyelitis and wound care in an eastern black rhinoceros (Diceros bicornis michaeli)

A 14-yr-old female eastern black rhinoceros (Diceros bicornis michaeli) presented with progressive suppurative osteomyelitis in her left hind lateral toe. beta-Hemolytic Streptococcus sp. was isolated. The animal was treated with multiple systemic antibiotics, and topical wound cleansing. Repeated debridements and nail trimmings were performed for 5 mo prior to electing amputation. The toe was surgically amputated under general anesthesia between the first and second phalanges. Analgesia was diffused into the wound topically via a catheter and elastomeric pump. The open amputation site was covered with adherent drapes and a negative-pressure wound therapy device provided vacuum-assisted closure (V.A.C.) for 72 hr. Three months later this animal developed a deep dermal ulcer on the lateral aspect of the right hind limb, at the level of the stifle. Methicillin-resistant Staphylococcus aureus was isolated. The wound was managed by initial daily lavage, followed by 1 mo of V.A.C. therapy, with 72 hr between dressing changes. Clinically, this therapy expedited the formation of healthy granulation tissue and overall healing was accelerated. The animal tolerated the machine and bandage changes well via operant conditioning. The use of negative-pressure wound therapy appeared to shorten time to resolution of slow-healing wounds in black rhinoceros.