A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.Major pathogens involved in fish farm salmonid infections include the gram negative species Flavobacterium psychrophilum, Yersinia ruckeri, and Aeromonas salmonicida. They are the etiological agents of cold water disease, enteric red mouth disease, and furunculosis, respectively. These pathologies are common worldwide and produce considerable economic losses in the fish farming industry.Cold water disease particularly affects juvenile fish (3), and the causal agent is F. psychrophilum, a fastidious bacterium that is difficult to grow (12). The bacterium causes saddle-like external lesions near the dorsal fin and it can also be found in the mouth, spleen, and brain tissues. The fish may darken and develop bacteremia with the microorganism present throughout the animal. Y. ruckeri is an important pathogen in intensive aquaculture of trout and salmon. Disease outbreaks are related to stress (4), and little information is available about the biology of the bacterium. Infected fish present characteristic red eyes and mouth as well as internal hemorrhages. Diagnostic methods include culturing, serology, and molecular biology techniques (5, 9, 16). A. salmonicida also produces an infection in fish that causes muscle lesions which could produce ulcers on the surface of the skin and lead to septicemia. Different diagnostic methods have been developed, including enzymelinked immunosorbent assays (19,20), agglutination tests (11), and PCR probes (14).On the other hand, an efficient selective medium for reducing the growth of the background flora and facilitating the isolation and identification of each of these three bacteria species remains to be described. To combat these infections, vaccination has been shown to be an effective method for the prevention of enteric red mouth disease (17) and, less efficiently, for furunculosis (10, 13), but there is no effective control for cold water disease. In most cases, antimicrobial compounds should be used to control disease outbreaks caused by any of these microorganisms. Thus, a rapid and effective diagnostic method is essential for the application of specific treatment.Although multiplex PCR (m-PCR) has been widely applied to the detection of multiple viruses and bacteria in clinical specimens (2, 6, 7), it has not been applied to the detection of fish pathogens (1, 15). In th...

Bovine tuberculosis (TB) infection is infrequently diagnosed in sheep. Most reports are from single individual cases or flock outbreaks. However, in Spain several outbreaks have been reported recently, all of which had epidemiological links with TB-infected cattle herds. A total of 897 sheep suspected of being infected with TB and belonging to 23 flocks cohabiting with TB-infected cattle herds and/or goats were tested between 2009 and 2013 in Galicia (north-western Spain), using pathological, immunological and molecular techniques. Of these, 50.44% were positive by culture, 83.23% by histopathology and 24.92%, 4.86% and 59.42% by single intradermal tuberculin test (SITT), interferon-γ and ELISA, respectively. Results suggest that in circumstances akin to those in our study, sheep may be considered as a potential source of TB. We conclude that under similar conditions, serious consideration should be given to TB testing sheep, as they may represent a potential risk to other susceptible co-habiting species. The SITT and ELISA are recommended as the simplest and most cost-effective initial approaches for the diagnosis of TB in sheep under field conditions. However, when possible, interferon-γ should be applied to increase sensitivity.

We describe the pathology, isolation and characterisation of a virus responsible for an outbreak of a systemic haemorrhagic disease causing high mortality in tadpoles of the common midwife toad Alytes obstetricans in the 'Picos de Europa' National Park in northern Spain. The virus, provisionally designated as the common midwife toad virus (CMTV), was isolated from homogenates of visceral tissue from diseased toad tadpoles following inoculation on epithelioma papulosum cyprini (EPC) cells. Molecular characterisation of the virus, including sequence analysis of the DNA polymerase and major capsid protein genes, showed that the isolated virus was a ranavirus with marked sequence identity to other members of the genus Ranavirus. A rabbit antiserum raised against purified virions was prepared and used to definitively demonstrate systemic distribution of the virus in diseased tadpoles, indicating that the isolated virus was the primary pathogen.

This report describes the isolation and characterisation of the common midwife toad virus (CMTV) from juvenile alpine newts (Mesotriton alpestris cyreni) and common midwife toad (CMT) tadpoles (Alytes obstetricans) in the Picos de Europa National Park in Northern Spain in August 2008. A comparative pathological and immunohistochemical study was carried out using anti-CMTV polyclonal serum. In the kidneys, glomeruli had the most severe histological lesions in CMT tadpoles, while both glomeruli and renal tubular epithelial cells exhibited foci of necrosis in juvenile alpine newts. Viral antigens were detected by immunohistochemical labelling mainly in the kidneys of CMT tadpoles and in ganglia of juvenile alpine newts. This is the first report of ranavirus infection in the alpine newt, the second known species to be affected by CMTV in the past 2 years.

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