G. Suarez

Mycobacterium tuberculosis subsp. caprae subsp. nov.: A taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain

Isolates from the Mycobacterium tuberculosis complex cultured from caprine pathological tissue samples were biochemically and genetically characterized. The isolates were negative for nitrate reduction and niacin accumulation, they weakly hydrolysed Tween 80, were sensitive to pyrazinamide (50 micrograms ml-1) and were resistant to 1 and 2 micrograms tiophene-2-carboxylic acid hydrazide ml-1 but not to 5 or 10 micrograms tiophene-2-carboxylic acid hydrazide ml-1. Sequencing of the pncA gene revealed a polymorphism characteristic of M. tuberculosis, whereas oxyR, katG and gyrA sequences were characteristic of Mycobacterium bovis. The fingerprinting patterns obtained with IS6110, direct repeats and polymorphic G+C-rich sequence-associated RFLP and direct variable repeat-spacer oligonucelotide typing (spoligotyping) segregated these isolates from the other members of the complex. The results of this testing, together with the repeated association of this micro-organism with goats, suggest that a new member of this taxonomic complex not matching any of the classical species had been identified. This unusual mycobacterium may play a role in the epidemiology of animal and human tuberculosis in Spain. The name Mycobacterium tuberculosis subsp. caprae subsp. nov. is proposed for these isolates. The type strain of Mycobacterium tuberculosis subsp. caprae subsp. nov. is gM-1T (= CIP 105776T).

Staphylococcus aureus subsp. anaerobius subsp. nov., the Causal Agent of Abscess Disease of Sheep

A new subspecies, Staphylococcus aureus subsp. anaerobius, is described on the basis of a study of 84 strains isolated from young sheep affected by the so-called “abscess disease.” The strains of this new subspecies grow well under anaerobic conditions, but not at all or only very weakly under aerobic conditions. They are catalase and benzidine negative and form small unpigmented colonies. Anaerobically they produce L-lactic acid from glucose. The chemical composition of the cell wall, the results of deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments, and the immunological relationships among L-lactate dehydrogenases demonstrated that these strains are closely related to Staphylococcus aureus. The type strain is strain MVF-7(= ATCC 35844).

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).

In vitro infection of cells of the monocytic/macrophage lineage with bovine leukaemia virus.

The oncogenic retrovirus bovine leukaemia virus (BLV) primarily infects B cells. Most infected animals remain asymptomatic for long periods of time before an increase in circulating B cells or localized tumours can be observed. This long clinical latency period may be explained by cells of the monocyte/macrophage lineage (M/M) becoming infected and acting as a reservoir for the virus, as shown for other retroviruses (human immunodeficiency virus-1, feline immunodeficiency virus). M/M cells in different stages of differentiation (HL-60, THP-1, U-937, J774, BGM, PM2, primary macrophages of sheep and cows) were cultured with BLV produced by permanently infected donor cells (FLKBLV and BLV-bat(2)). Donor cells were inhibited from multiplying by either irradiation or treatment with mitomycin C. In other experiments, supernatant from donor cells containing virus was used. In co-culture with the donor cells, the less differentiated monocytic cells showed severe cellular changes such as differentiation, vacuolization, cell lysis and membrane blebbing; apoptosis was a frequent phenomenon. Budding and extracellular viruses were also observed. The more differentiated macrophage cells, although they showed less signs of infection by microscopy, had a complete BLV protein profile, as seen by Western blotting; bands corresponding to p24CA (Gag) and its precursors were clearly seen. In addition, gp51SU was identified by syncytia formation assays. It is concluded that M/M cells may be infected by BLV, the consequences of the infection differing according to the type of cell.

Determination of patulin by reversed-phase high-performance liquid chromatography with extraction by diphasic dialysis

A simple and economical method has been developed for the determination of patulin in apple juice. The sample is extracted with ethyl acetate in a diphasic dialysis system, and the extract is cleaned up by elution from a Sep-Pak cartridge. Patulin is detected and determined by reversed-phase high-performance liquid chromatography using a Novapak C18 column and an ultraviolet detector. The lower detection limit is 1 microgram l-1 and the recovery is 85% at the 20 micrograms l-1 level.

Viability of Listeria monocytogenes in Milk Treated with Hydrogen Peroxide

The susceptibility of Listeria monocytogenes to hydrogen peroxide in sterilized and raw milk was studied. In raw milk, L. monocytogenes was less susceptible to H2O2 than milk microflora. The ratio of L. monocytogenes to total milk micro-organisms (natural microflora plus added L. monocytogenes ) increased when raw milk was stored at refrigeration temperature (4°C), due to a selective enrichment of Listeria present in milk. In sterilized milk, a concentration of 0.0495% H2O2 and 9 h were required to produce complete destruction of L. monocytogenes when this microorganism was in pure culture, although a reduction in listeria counts was observed at 1.5 h. When sterilized milk was simultaneously contaminated with L. monocytogenes , Staphylococcus aureus and Streptococcus faecalis , a decrease in L. monocytogenes count during the first 24 h was observed at 0.0495% H2O2. From this time L. monocytogenes recovered and multiplied reaching levels similar to the initial counts at the end of the experiment.

Identification of coagulase negative staphylococci isolated from lambs as Staphylococcus caseolyticus.

A group of 17 strains of coagulase negative staphylococci isolated from slaughtered lambs, and which could not be identified with the conventional methods, exhibited high levels of DNA homology (92%) with the S. caseolyticus reference strain. The isolates described in this study provide a more extensive comprehension of S. caseolyticus. The original description of this species was based on only two strains isolated from milk. To our knowledge, S. caseolyticus had never been previously associated with animal microflora.

Enterotoxin production by strains of Staphylococcus intermedius and Staphylococcus aureus isolated from dog infections.

Sixty-six strains of S. intermedius and 10 of S. aureus isolated from infected dogs were examined for enterotoxin production. 39.5% of the strain (37.9% of S. intermedius and 50% of S. aureus) produced one or more enterotoxins. The predominant types produced by S. intermedius were C1 and C2, and only two of the strains synthesized enterotoxin A. One of the S. aureus strains produced the toxic shock syndrome toxin 1.

Fate of Listeria monocytogenes during manufacture and ripening of semi‐hard cheese

Semi‐hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 105Listeria monocytogenes/ml in cheeses 1 and 2 and 4 times 103L. monocytogenes/ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.