J.M. Alonso

Serotypes, Virulence Genes, and Intimin Types of Shiga Toxin (Verotoxin)-Producing Escherichia coli Isolates from Healthy Sheep in Spain

ABSTRACT Fecal swabs obtained from 1,300 healthy lambs in 93 flocks in Spain in 1997 were examined for Shiga toxin-producing Escherichia coli (STEC). STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157 STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC strains were characterized in this study. PCR showed that 213 (55%) strains carried the stx1 gene, 10 (3%) possessed the stx2 gene, and 161 (42%) carried both the stx1 and the stx2 genes. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains, respectively. The STEC strains belonged to 35 O serogroups and 64 O:H serotypes (including 18 new serotypes). However, 72% were of 1 of the following 12 serotypes: O5:H−, O6:H10, O91:H−, O117:H−, O128:H−, O128:H2, O136:H20, O146:H8, O146:H21, O156:H−, O166:H28, and ONT:H21 (where NT is nontypeable). Although the 384 STEC strains belonged to 95 different seropathotypes (associations between serotypes and virulence genes), 49% of strains belonged to only 11. O91:H− stx1stx2 (54 strains) was the most common seropathotype, followed by O128:H− stx1stx2 (33 strains) and O6:H10 stx1 (25 strains). Three strains of serotypes O26:H11, O156:H11, and OX177:H11 had intimin type β1; 5 strains of serotype O157:H7 possessed intimin type γ1; and 15 strains of serotypes O49:H−, O52:H12, O156:H− (12 strains), and O156:H25 had the new intimin, intimin type ζ. The majority (82%) of ovine STEC strains belonged to serotypes previously found to be associated with human STEC strains, and 51% belonged to serotypes associated with STEC strains isolated from patients with hemolytic-uremic syndrome. Thus, this study confirms that healthy sheep are a major reservoir of STEC strains pathogenic for humans.

Serotypes, phage types and virulence genes of Shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.

Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans.

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H– [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H–, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H–, O6:H10, O91:H–, O117:H–, O128:H–, O128:H2, O146:H8, O146:H21, O156:H–, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.

Detection and characterisation of O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in wild boars

The aim of this work was to determine the prevalence and characteristics of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) in free-ranging wild boars killed during the hunting season in southwest Spain. Faecal samples from 212 wild boars (Sus scrofa) were collected and examined for STEC. Characterisation of isolates was performed by PCR, serotyping, phage typing, and pulsed-field gel electrophoresis (PFGE). E. coli O157:H7 and non-O157 STEC were isolated from 7 (3.3%) and 11 (5.2%) animals, respectively, and the resulting 19 isolates were characterised. The PCR procedure indicated that 4 isolates carried the stx(1) gene, 12 carried the stx(2) gene, and 1 contained both of these genes. The ehxA, eae, and saa genes were detected in 13, 8, and 1 of the isolates, respectively. The eae-positive isolates comprised the types eae-gamma 1 and eae-zeta. The isolates belonged to 11 O:H serotypes, including 4 new serotypes not previously reported within STEC strains, and the majority of them were from serotypes previously associated with human infection. E. coli O157:H7 isolates belonged to phage types associated with severe human illness: PT14, PT34, and PT54. Indistinguishable PFGE types were found in E. coli O157:H7 isolates recovered from a wild boar and from a human patient with diarrhoea living in the same geographic area.

Pheno-genotypic characterisation of Escherichia coli O157:H7 isolates from domestic and wild ruminants.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with ruminants recognised as their main natural reservoir. The aim of this work was to determine the phenotypic features and genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle and deer faeces and from unpasteurised goat milk in Spain over a period of 11 years. Characterisation was performed by polymerase chain reaction (PCR), phage typing and pulsed-field gel electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting and beta-glucuronidase positive) originating from deer faeces was detected. Genes encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying only the stx(2) gene. The isolates were from nine different phage types, although 67.4% were restricted to only three: PT14, PT34 and PT54. PT54 was the most prevalent phage type and contained isolates from cattle, sheep and deer. Majority of the isolates were from phage types previously found in strains associated with human infection. XbaI-PFGE identified 33 different types and 11 groups of closely related types (more than 85% similarity), one of which included 21 (45.7%) isolates originating from different animal species, including deer. These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates and contribute to earlier investigations identifying deer as a natural source of E. coli O157:H7. The study also highlights the emergence of phenotypic variants of E. coli O157:H7, which may not be identified by routine culture methods or by biochemical tests used to characterise serotype O157:H7.

Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2

Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection.

Enzymatic activities of Dermatophilus congolensis measured by API ZYM

API ZYM kit was used to test enzymatic activities on eighteen strains of Dermatophilus congolensis. All strains produced lipase and acid phosphatase, which act on lipids, and leucine arylamidase which act on proteins. Another 10 exoenzymes were present in at least one of the strains.

AETIOLOGY OF CAPRINE CONTAGIOUS AGALACTIA SYNDROME IN EXTREMADURA, SPAIN

ing fish were treated by adding sodium benzylpenicillin DAVIS, H. S. (1922) A new gill disease in trout. Transactions of the American (10,000 iu/litre) to the water for 24 hours. Two days later, the Fisheries Society 56, 156-159 antibiotic treatment was repeated. Following these meaDECOSTERE, A. (1997) Development of a gill perfusion model and consesurements, a marked drop in mortality was recorded. The quent study of the adherence of Flavobacterium columnare to the gill tissue. fish appeared to be much more active and skin lesions MSc thesis, Stirling, Scotland diminishapped. hmoeativeandskinlesons DECOSTERE, A., HAESEBROUCK, F. & DEVRIESE, L. A. (1997) Shieh medium supplemented with tobramycin for the selective isolation of F columnare was first described by Davis in 1922. Since Flavobacterium columnare (Flexibactercolumnaris) from diseased fish.Journal then, it has been stated that all freshwater fishes are susceptiof Clinical Microbiology 35, 322-324 ble to columnaris under environmental conditions favourable DECOSTERE, A., HAESEBROUCK, F., TURNBULL, J. F. & CHARLIER, G. to the bacterium and stressful to the fish (Dalsgaard 1993, (1998) Influence of environmental parameters on the adhesion of high and Wakabayashi 1993). In this case, the high level of nitrite most low virulence Flavobacterium columnare strains to isolated gill arches. Journal likely triggered the columnaris outbreak, as was found by ofFish Diseases 21, 1-11 Hanson and Grizzle (1985) in channel catfish (IctaluruspuncHANSON, L. A. & GRIZZLE, J. M. (1985) Nitrite-induced predisposition of adhesion ofF columnare to the ill tissue is indeed channel catfish to bacterial diseases. Progressive Fish Culturist 47, 98-101 tatus). Thne aaneslon or r columnare to tne glll lssueSHIEH, H. S. (1980) Studies on the nutrition of a fish pathogen, Flexibacter enhanced in the presence of nitrite (Decostere 1997, columnaris. Microbios letters 13, 129-133 Decostere and others 1998). TOMEY, W. A. (1985) Ziekten bij aquariumvissen. Best, Nederland, Zuid F columnare does not grow on blood agar. Special media Boekprodukties b.v. p 140 such as Cytophaga (Anacker and Ordal 1959) or Shieh WAKABAYASHI,H. (1993) Bacterial diseases of fish. EdsV. Inglis, R. J. Roberts, (1980) medium are needed for isolation. This explains why N. R. Bromage. Blackwell Scientific Publications, Oxford. p 23-33 columnaris disease frequently is misdiagnosed. Another reason is that columnaris disease resembles a parasitic (costiasis, white spot) and a fungal infection (saprolegniosis). In all cases, the presence of white-grey areas on the skin is evident. However, in columnaris disease, these areas are typically localised on the head and cranial dorsal part of the body and around the dorsal fin. The lesions are mostly circular as if o g of c p spreading from a single focus in all directions at the same Aetiology Of CaPrine rate. Differential diagnosis can easily be made by means of* microscopic examination of skin scrapings of the affected con iou ag-alact areas. In the case described, effective treatment consisted of two syndrome In parts: first, elimination of the predisposing factor, that is, decreasing the nitrite level; and, secondly, antibiotic treatExtremad ura, Spain ment. Sodium benzylpenicillin appeared to be effective, as was reported previously by Bassleer (1983) and Tomey (1985). This therapeutic agent is relatively cheap and does M. C. Gii, M. HERMOSO DE MENDOZA, not colour the water. Water change has to be complete after J. REY, J. M. ALONSO, J. B. POVEDA, 24 hours, since sodium benzylpenicillin might produce substances toxic for fish after this time (Bassleer 1983). Other J HERMOSO DE MENDOZA antimicrobial agents can be used to treat columnaris disease, both in the water and the feed. An excellent review has been CONTAGIOUS agalactia (CA) ofgoats and sheep is one of the provided by Amend (1970). most serious diseases affecting small ruminants. Endemic in In conclusion, the risk of a severe columnaris outbreak most Mediterranean countries (Lambert 1987), the disease is not a problem to be taken lightly, especially when dealing is widely distributed throughout most of Spain (Garrido and with live-bearing fish. Since F columnare can cause heavy others 1987, Real and others 1994). The disease in goats may mortalities over a short period of time, the condition must be be caused by several Mycoplasma species (DaMassa and othdiagnosed quickly and measures taken accordingly. ers 1992); this short communication describes the numbers Veterinary Record (1999) and types of mycoplasma isolated from goats affected by CA 144,24-25 in the Extremadura region of Spain. ACKNOWLEDGEMENTS This study was carried out between 1991 and 1995 in 43 M. C. Gil, PhD, goat flocks affected by CA outbreaks in Extremadura, southM. Hermoso de Mendoza, The authors appreciate the cooperation of the farmer conwest Spain. Five hundred and ninety-six adult and young PhD, cerned. The Fund for Scientific Research, Flanders, is goats, mainly of the Serrana breed, were sampled and a total J. Rey, PhD, acknowledged for providing a grant to A. D. of 1309 specimens were collected from both live and, after J. M. Alonso, PhD, routine necropsy, dead animals. Mycoplasmas were isolated J. Hermoso de Mendoza,

A zoonotic ringworm outbreak caused by a dysgonic strain of Microsporum canis from stray cats

BACKGROUND Cats are frequent carriers of Microsporum canis and veterinary students are at high risk of exposure and acquisition of the organism a la infección. OBJECTIVES An outbreak of zoonotic ringworm carried by a litter of stray cats is described. Four veterinary students, four dogs, and six cats living in five separate locations were affected. All had direct or indirect contact with the infected kitten litter. We tried to identify the causal dermatophyte. METHODS Conventional and mycological culture methods were used. RESULTS Microscopic features of scrapings and hairs treated with 20% KOH strongly suggested a M. canis etiology, and a diagnosis of ringworm was empirically supported by successful treatment of humans and animals. Nevertheless, cultures failed to show the expected morphology. CONCLUSIONS Culture features of our strain are compared with those described by other authors for dysgonic M. canis strains. Epidemiological features are also discussed.

Longitudinal study of Shiga toxin-producing Escherichia coli shedding in sheep feces: persistence of specific clones in sheep flocks.

ABSTRACT To provide information on the persistence and maintenance of colonization with Shiga toxin-producing Escherichia coli (STEC) in sheep, pulsed-field gel electrophoresis analysis of STEC isolates (n = 145) belonging to serogroups O5, O91, and O146 from 39 healthy animals was performed in a 12-month longitudinal study carried out with four sheep flocks. At the flock level as well as the individual-animal level, the same clones were obtained on sampling occasions separated by as much as 11 months.