J. F. Fernández-Garayzábal

Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish

ABSTRACT A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

Winter disease outbreak in sea bream (Sparus aurata) associated with Pseudomonas anguilliseptica infection

Abstract The nature of an outbreak of ‘winter disease’ affecting both juvenile and adult sea-bream ( Sparus aurata ) in several Iberian Peninsula farms from January to April of 1996 is described. The average mortality rate was approximately 10–15%, although in some fish farms mortality reached 30%. Pure cultures of aerobic Gram-negative filamentous rods were isolated from the kidney of diseased fish, as well as from some of the liver and ascitic fluid samples. 16S rRNA gene sequence analysis identified the isolates from diseased fish as Pseudomonas anguilliseptica . The results of biochemical and physiological tests of clinical isolates and the type strain of P. anguilliseptica were entirely consistent with the genotypic identification. The isolation of P. anguilliseptica exclusively from the diseased fish of all affected fish farms, together with the failure to isolate this bacterium from non-affected fish of the same farms, suggest that P. anguilliseptica is likely to be the agent responsible for the ‘winter disease’ outbreak in the sea-bream examined. The relationship of the ‘winter disease’ outbreaks to stressful environmental conditions is discussed.

Prevalence and aetiology of subclinical mastitis in dairy ewes of the Madrid region

A bacteriological survey for studying the prevalence and aetiology of subclinical mastitis was carried out in 22 dairy sheep flocks of the Madrid region. A total of 1128 milk samples were collected from 564 ewes. 17 Manchega flocks (5 flocks had mechanical milking and 12 flocks had hand milking) and 5 Assaf flocks (all had mechanical milking) were included in the study. Prevalence of subclinical mastitis in the different flocks ranged from 4.5–67% of the glands and 9–83% of the animals, with an average of 21 and 34.%, respectively. Prevalence of subclinical mastitis in Manchega flocks with mechanical milking was lower than those with hand milking (P<0.05). Assaf flocks had higher prevalence rates than Manchega flocks with mechanical milking (P<0.05). Increase of subclinical mastitis as lactation proceeded was observed only in Assaf ewes. Prevalence of subclinical mastitis was lower in primiparous Manchega and Assaf ewes than those which had two of more lactations. CMT score 1(+), with the best combination of sensitivity and specificity values, can be recommended as a threshold value for detecting subclinical mastits. Coagulase-negative staphylococci were the most prevalent bacteria, representing 68% of the isolates. Staphylococcus epidermidis (40%), was the most prevalent species followed by Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Corynebacterium was the second bacterial group in importance according with the distribution among flocks, being isolated from 41% of the flocks and representing 10% of the isolates, which suggests for these bacteria, a clinical significance higher than that traditionally considered as responsible for subclinical mastitis in sheep. Subclinical mastitis seems to be, as deduced from the high prevalence observed in this study, an important health problem for milking sheep flocks in the Madrid region.

Corynebacterium mastitidis sp. nov., isolated from milk of sheep with subclinical mastitis.

Fourteen strains of a hitherto unknown catalase-positive, aerobic, gram-positive coryneformlike organism were isolated from the milk of sheep with subclinical mastitis from different regions of Spain. The strains phenotypically closely resembled one another and biochemically were similar to Corynebacterium urealyticum and Corynebacterium afermentans subsp. lipophilum. The results of chemotaxonomic investigations were consistent with membership in the genus Corynebacterium, and comparative 16S rRNA gene sequencing studies showed that the unknown bacterium from sheep was indeed a member of the genus Corynebacterium. Within the genus Corynebacterium the new bacterium formed a distinct subline that exhibited > 4% sequence divergence with other species. Based on both phenotypic and phylogenetic findings, a new species, Corynebacterium mastitidis, is proposed for the organisms from mastitic sheep. The type strain of C. mastitidis is CECT 4843 (= S-8).

Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and Human Listeria monocytogenes Isolates

ABSTRACT A total of 153 strains of Listeria monocytogenesisolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains ofL. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride‐phenylethanol‐moxalactam, listeria selective medium–Oxford formulation, polymyxin‐acriflavine‐lithium chloride‐ceftazidime‐aesculin‐mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).

Phenotypic and phylogenetic characterization of some unknown coryneform bacteria isolated from bovine blood and milk: description of Sanguibacter gen.nov.

16S rRNA gene sequencing studies were performed on some Gram‐positive coryneform bacteria of unknown taxonomic position isolated from blood and milk of healthy cows. Comparative sequence analysis demonstrated that the milk isolates corresponded to Oerskovia xanthineolytica whereas those from blood consisted of two distinct, albeit highly related species, within the family Cellulomonadaceae. Based on the phylogenetic and phenotypic distinctiveness of the blood isolates, it is proposed that they be classified in a new genus Sanguibacter.

Corynebacterium camporealensis sp. nov., associated with subclinical mastitis in sheep

Four strains of a hitherto-unknown catalase-positive, facultatively anaerobic Corynebacterium species were isolated from the milk of sheep affected by subclinical mastitis. The most characteristic phenotypic reactions of the four strains were their weak fermentative acid production from glucose, their failure to produce acid from mannitol, xylose, sucrose and maltose, and a strong CAMP reaction with Staphylococcus aureus. Chemotaxonomic investigations revealed the presence of a cell wall based on meso-diaminopimelic acid and short-chain mycolic acids, which is consistent with the genus Corynebacterium. A comparative 16S rRNA gene sequence analysis confirmed that the organisms from sheep were members of the genus Corynebacterium, where they formed a distinct subline, exhibiting > 4% sequence divergence with other known Corynebacterium species. Based on both phenotypic and phylogenetic findings, a new species, Corynebacterium camporealensis, is proposed. The type strain of Corynebacterium camporealensis is CECT 4897 (= CRS-51T).

Mycobacterium avium subspecies paratuberculosis in fallow deer and wild boar in Spain

Mycobacterium avium subspecies paratuberculosis is the aetiological agent of a chronic granulomatous enteritis in ruminants known as Johne’s disease or paratuberculosis. It is considered to be one of the most serious diseases affecting dairy cattle worldwide, being responsible for significant economic losses (Harris and Barletta 2001). Its clinical relevance is commonly recognised in captive or farmed wild ruminants, but some studies have suggested that wild ruminant populations could also be an important natural reservoir of M avium subspecies paratuberculosis. The pathogen has already been isolated from a number of wild ruminant species, such as red deer (Cervus elaphus), fallow deer (Dama dama) and tule elk (Cervus elaphus nannodes) (Cook and others 1997, Pavlik and others 2000). In Spain, there has been only one study of the presence of the pathogen in free-ranging fallow deer (Marco and others 2002). However, the host range of M avium subspecies paratuberculosis is not limited to ruminants, and it has also been isolated from a wide variety of wild non-ruminant species, such as fox (Vulpes vulpes), stoat (Mustela erminea), weasel (Mustela nivalis), crow (Corvus corone), rook (Corvus frugilegus), jackdaw (Corvus monedula), rat, wood mouse (Apodemus sylvaticus), rabbit (Oryctolagus cuniculus), hare (Lepus capensis) and badger (Meles meles) (Beard and others 2001). There have been very few studies on the presence of the pathogen in wild boar (Sus scrofa), and none in Spain, despite the fact this species is known to be a relevant reservoir of another important and related pathogen, Mycobacterium bovis (Aranaz and others 2004). This short communication describes a study to investigate the presence of M avium subspecies paratuberculosis in wild red deer, fallow deer and wild boar in the south and west of Spain. A total of 260 animals (101 red deer, 94 fallow deer and 65 wild boar) was examined for the presence of M avium subspecies paratuberculosis between 2001 and 2003. All animals had been hunted; no clinical signs of disease were observed and no gross lesions were detected at postmortem examination. Samples of intestine, ileocaecal valve and mesenteric lymph nodes were collected under aseptic conditions and kept refrigerated at 4°C until they were processed in the laboratory within eight hours. The tissue specimens from each animal were pooled and processed for M avium subspecies paratuberculosis isolation as described by Greig and others (1999). Selective media (Middlebrook 7H11 agar, Herrold’s egg yolk medium [HEYM], HEYM plus sodium pyruvate and Lowestein-Jensen medium) (Biomedics) supplemented with antimicrobials (Mycobacteria Selectatab; MAST Laboratories) and 2 mg/litre mycobactin J (Allied Monitor) were incubated aerobically at 37°C and checked monthly for up to six months for the presence of colonies compatible with M avium subspecies paratuberculosis. Identification of suspect colonies was confirmed by two PCR reactions, the first aimed at the M avium subspecies paratuberculosis-specific insertion sequence IS900 (Millar and others 1995), and the second targeting a novel DNA sequence present in M avium subspecies paratuberculosis isolates but absent in other strains from the M avium-Mycobacterium intracellulare complex (Collins and others 2002). M avium subspecies paratuberculosis was isolated from one wild boar and one fallow deer from the same area. No positive cultures were obtained from the red deer. Both isolates belonged to the cattle type, which is the most common in Europe regardless of the animal source (Whittington and others 2000). The low frequency of detection obtained in the fallow deer (1·1 per cent) was similar to that reported in the north of Spain by Marco and others (2002), as well as in other areas of Europe such as the Czech Republic (Pavlik and others 2000) or Scotland (Fawcett and others 1995). This suggests that, unlike farmed animals, free-ranging wild cervid populations are rarely infected by the pathogen (Jessup and Williams 1999). The present study is the first of wild boar carried out in Spain and the second one in Europe. Machackova and others (2003) investigated the prevalence of M avium subspecies paratuberculosis in wild boar in several central European countries (Bosnia and Herzegovina, Croatia, the Czech Republic, Hungary and Slovakia). The detection frequency of the pathogen reported by those authors was even lower than that found in the present study (1·5 per cent with a confidence interval of 95 per cent). From these data, it seems likely that a low prevalence of M avium subspecies paratuberculosis infections in wild boar should also be expected. Despite low frequencies of detection found in the three animal species under study, the epidemiological consequences should be considered with caution. It is well known that most infected dairy cows do not develop clinical signs despite shedding the pathogen in their faeces (Chiodini and others 1984). These infected animals represent a source of contamination of the environment, where the pathogen is able to persist for long periods of time (Larsen and others 1956), contributing to the infection of new hosts by the faecal-oral route (Chiodini and others 1984). A similar situation could also occur in wild deer and boar populations. Thus, the detection of deer and boar infected by M avium subspecies paratuberculosis is still epidemiologically relevant, as these animals might contribute to the persistence and spread of the pathogen, particularly where higher densities of animals occur (such as at drinking points and in dens and shelters). In particular, wild boar should be given greater consideration, as they have the potential to spread the pathogen in a broader range due to their active behaviour, high mobility and increasing populations in some areas.

Methicillin resistant Staphylococcus aureus (MRSA) carriage in different free-living wild animal species in Spain

Methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening pathogen in humans and its presence in animals is a public health concern. The aim of this study was to measure the prevalence of MRSA in free-living wild animals. Samples from red deer (n=273), Iberian ibex (n=212), Eurasian Griffon vulture (n=40) and wild boar (n=817) taken from different areas in Spain between June 2008 and November 2011 were analyzed. Characterization of the isolates was performed by spa typing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing. A low prevalence of MRSA was found with 13 isolates obtained from 12 animals (0.89%; 95% CI: 0.46-1.56). All MRSA sequence types belonged to ST398 (t011 and t1451) and ST1 (t127). Genotypes and antimicrobial susceptibility patterns (tetracycline resistance in ST398 and clindamycin-erythromycin-tetracycline resistance in ST1) suggest that the MRSA found probably originated in livestock (ST398) or humans (ST1). This is the first report of MRSA carriers in free-living wild animals in Europe. Although our data showed that MRSA prevalence is currently low, free-living wild animals might act as reservoir and represent a potential risk for human health.