Ana I. Vela

Bovine Tuberculosis (Mycobacterium bovis) in Wildlife in Spain

ABSTRACT Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

Genotyping of Francisella tularensis Strains by Pulsed-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism Fingerprinting, and 16S rRNA Gene Sequencing

ABSTRACT We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Analysis of Genetic Diversity of Streptococcus suis Clinical Isolates from Pigs in Spain by Pulsed-Field Gel Electrophoresis

ABSTRACT Pulsed-field gel electrophoresis (PFGE) was used to investigate the diversity of Streptococcus suis isolates of various serotypes recovered from swine clinical samples in Spain. Capsular types 9 (64.9%) and 2 (14.8%) were the most frequently isolated serotypes followed by serotype 7 (5.9%) and serotype 8 (4.3%). The PFGE results of this study with 60 different pulsotypes indicate a great genetic diversity among the S. suis isolates, which is consistent with the broad distribution of S. suis in the swine population. Forty-five percent of the pulsotypes corresponded to single isolates, no pulsotype was common to all farms, and at least 3 different pulsotypes were isolated in 56% of herds in which more than 3 clinical isolates were analyzed. These results reveal a great diversity both between and within herds throughout the strains of S. suis studied, demonstrating that different strains of S. suis are associated with infection in pigs. Some pulsotypes were more frequently isolated and exhibited a wider distribution over herds than others, and were the unique or predominant strains in several herds, suggesting the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. Overall, the great genetic heterogeneity of the clinical strains of S. suis, the isolation of different strains within the same herd, and the predominance of particular strains in some herds are evidence that infection by S. suis is a dynamic process and reinforce the idea that the epidemiology of S. suis infection is very complex.

Unusual Outbreak of Clinical Mastitis in Dairy Sheep Caused by Streptococcus equi subsp. zooepidemicus

ABSTRACT This work describes an outbreak of clinical mastitis affecting 13 of 58 lactating ewes due to Streptococcus equi subsp. zooepidemicus. S. equi subsp. zooepidemicus was isolated in pure culture from all milk samples. All the clinical isolates had identical biochemical profiles and antimicrobial susceptibility patterns and also exhibited indistinguishable macrorestriction patterns by pulsed-field gel electrophoresis, indicating that all cases of mastitis were produced by a single strain.

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.

Clonal diversity of Staphylococcus aureus originating from the small ruminants goats and sheep.

Staphylococcus aureus is an important pathogen in humans and many animal species. The prevalence of different clonal types in animal species remains largely unknown. We analyzed 267 S. aureus from intramammary infections in goats (47) and sheep (220) by spa typing, multi-locus sequence typing (MLST) and antimicrobial susceptibility. The most frequent spa types in goats were t337 (N=9), t759 (N=6) and t1534 (N=5). Sheep isolates mainly belonged to spa types t1534 (N=72), t2678 (N=29) and t3576 (N=20). Eighteen novel spa-types were observed; two from goat strains, 13 from sheep and three in both species. The majority of the goat strains grouped in MLST CC133 (N=10) and ST522 (N=10), followed by CC9 (N=9), while the majority of the sheep strains were of ST522 (N=108) followed by CC133 (N=86) and CC130 (N=11). Nine new MLST types were detected; three in goat and sheep isolates (ST1739, ST1758 and ST1780), two identified in goats only (ST1740 and ST2061) and four in sheep only (ST1742, ST1743, ST1781 and ST2011). Strains showed resistance below 20% against penicillin and tetracycline; a strong association between CC-types and penicillin resistance was observed. No resistance was detected to cefoxitin, quinupristin-dalfopristin, rifampicin and vancomycin. This study suggests that ST522 is the most common S. aureus clone associated with small ruminants followed by CC133.

Antimicrobial susceptibility of Listeria monocytogenes isolated from meningoencephalitis in sheep

The antimicrobial susceptibility to different antimicrobial agents of 41 Listeria monocytogenes strains isolated from sheep with meningoencephalitis and from feedstuff was tested by both microdilution and disk diffusion methods. Both sets of isolates of L. monocytogenes were susceptible to penicillin G, amoxicillin, cephalothin, erythromycin, vancomycin, rifampicin, gentamicin, kanamycin, trimethoprim, sulfisoxazole, chloramphenicol and ciprofloxacin, but resistant to tetracycline and doxycycline (7.3 and 4.9%, respectively). Tetracycline was the most frequent resistance trait in L. monocytogenes strains of animal origin. Four strains (9.8%) also exhibited reduced susceptibility (MIC 4 mg/l) to doxycycline suggesting the need of surveillance studies to monitor the antimicrobial resistance of Listeria strains of animal origin.

Reclassification of the members of the genus Tetrathiobacter Ghosh et al. 2005 to the genus Advenella Coenye et al. 2005.

The taxonomic position of the genera Advenella and Tetrathiobacter was examined. 16S rRNA gene sequence analysis revealed that the two genera are closely related, representing a monophyletic cluster with high sequence similarity (98.1-99.7%) within the family Alcaligenaceae. The phenotypic characteristics of the type strains of Advenella incenata, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis were re-examined using the API 20NE, API ZYM and API 50CH systems. Phylogenetic data together with similarities in phenotypic characteristics, G+C content and cellular acid composition suggest that they should be classified in the same genus. On the basis of the data presented, the two species of the genus Tetrathiobacter should be transferred to the genus Advenella, since this genus has nomenclatural priority. Therefore, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis should be transferred to the genus Advenella as Advenella kashmirensis comb. nov. (type strain WT001T=LMG 22695T=MTCC7002T) and Advenella mimigardefordensis comb. nov. (type strain DPN7T=DSM 17166T=LMG 22922T). Emended descriptions of Advenella incenata and the genus Advenella are also presented.

Streptococcus entericus sp. nov., isolated from cattle intestine

Biochemical, molecular chemical and molecular genetic studies were performed on an unknown gram-positive, catalase-negative, coccus-shaped organism isolated from the intestine of a cow affected with catarrhal enteritis. The organism was tentatively identified as a streptococcal species based on results of cellular morphological and biochemical tests. 16S rRNA gene sequencing studies confirmed its provisional identification as a member of the genus Streptococcus, but the organism did not correspond to any recognized species of this genus. The nearest phylogenetic relatives of the unknown coccus from a calf were Streptococcus acidominimus and Streptococcus suis. The unknown bacterium, however, was distinguished from these species and other animal streptococci by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus entericus sp. nov. The type strain is CECT 5353T (= CCUG 44616T).

Weissella ceti sp. nov., isolated from beaked whales (Mesoplodon bidens)

During an investigation into the microbiota of beaked whales (Mesoplodon bidens), nine isolates were obtained from different organs of four animals. The isolates were Gram-positive-staining, catalase-negative, short rod-shaped or coccoid organisms. A phylogenetic analysis based on 16S rRNA gene sequences of these isolates allocated them to the genus Weissella, showing 96.3 % and 96.0 % 16S rRNA gene sequence similarity with Weissella viridescens NRIC 1536(T)and Weissella minor NRIC 1625(T), respectively. On the basis of phenotypic, physiological and phylogenetic evidence, it is proposed that the new isolates from whales represent a novel species of the genus Weissella, Weissella ceti sp. nov. The type strain of Weissella ceti is 1119-1A-09(T) ( = CECT 7719(T) = CCUG 59653(T)).