Induction of ulcerative colitis in mice influences the course of infection with the nematode Trichuris muris.

The aim of this study was to assess the effect of infection with the nematode whipworm Trichuris muris on the course of chemically induced acute ulcerative colitis in CBA/J mice, a strain proven to be highly resistant to infection with T. muris. Each mouse was infected with 50 embryonated eggs of T. muris by oral gavage. Acute colitis was triggered by administering 4% dextran sulphate sodium (DSS) in the drinking water for nine consecutive days at different times after infection. Concurrent infection and DSS administration exacerbate the severity of the colitis while favouring the permanence of parasites in the intestine. The induction of ulcerative colitis from days 54 to 62 post-infection (p.i.), when all worms had been expelled, ameliorated the course of the inflammatory disease. When ulcerative colitis was triggered earlier on, from days 27 to 35 p.i., the beneficial effects on inflammatory events were clearly shown with signs of mucosal epithelization and regeneration as early as day 1 after DSS administration. Previous infections by T. muris therefore accelerate recovery from subsequently induced inflammatory bowel disease and such an effect assists the nematode to persist in the intestinal niche.

First Report of Cylindrocladiella parva and C. peruviana Associated with Black-foot Disease of Grapevine in Spain.

From 2007 to 2009, Cylindrocladiella-like isolates were recovered from grapevine (Vitis vinifera L.) roots with symptoms of black-foot disease in Spain, where the causal agents of this disease have been previously reported as Campylocarpon and Cylindrocarpon species (1,2). Three representative isolates were selected to confirm their identity: CPa1 and CPa2 from Asturias (northern Spain), and CPe523 from Cuenca (central Spain). Isolates were incubated on malt extract agar (MEA) and Spezieller Nährstoffarmer Agar (SNA) with carnation leaves (4) at 25°C for 10 days in darkness. On MEA, colonies developed light brown, cottony mycelium. On SNA, all three isolates produced chlamydospores in chains, and conidia were zero-to one-septate, but CPa1 and CPa2 produced longer conidia (10.4 to 18.9 [15.3] × 1.7 to 3.1 [2.4] µm) than CPe523 (6.4 to 12.3 [9.7] × 1.6 to 3.3 [2.4] µm). A fragment of the beta-tubulin gene from all isolates was sequenced with primers T1 and Bt2b (1) and deposited in GenBank (Accession Nos. JQ693133, JQ693134, and JQ693135). CPa1 and CPa2 showed high similarity (99%) to Cylindrocladiella parva (AY793486) and CPe523 showed high similarity (99%) to C. peruviana (AY793500), which is in agreement with the corresponding morphological features of these species (4). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seed soaked for 12 h in 300 ml of distilled water and autoclaved three times. Inoculum was prepared by inoculating two fungal disks (8 mm in diameter) of a 2-week-old culture of each isolate grown on potato dextrose agar to wheat seed and incubation at 25°C for 4 weeks. One-month-old grapevine seedlings were planted individually in 220-cc pots filled with a potting medium of sterilized peat moss and 10 g of inoculum, and grown in the greenhouse at 25°C in a completely randomized design. Controls were inoculated with sterile, noninoculated wheat seed. There were six replicate plants per isolate, with an equal number of controls, and the experiment was repeated once. Symptoms developed in all plants by 20 days post-inoculation and consisted of reduced vigor, necrotic root lesions, and occasionally mortality, all of which resembled the symptoms from grapevines in the field from which the isolates were originally recovered. Mean shoot dry weights of inoculated plants (0.25, 0.16, and 0.28 g for CPa1, Cpa2, and CPa523, respectively) were significantly lower (P < 0.05) than that of the controls (0.74 g). Mean root dry weights of inoculated plants (0.28, 0.16, and 0.29 g for CPa1, Cpa2, and CPa523, respectively) were also significantly lower (P < 0.05) than that of the controls (0.68 g). Isolates recovered from the roots of inoculated plants were identical morphologically and molecularly to C. parva and C. peruviana, thereby satisfying Koch's postulates. No symptoms were observed on the control plants. These Cylindrocladiella spp. have been reported from nurseries or vineyards in South Africa and New Zealand (3). To our knowledge, this is the first report of C. parva and C. peruviana associated with black-foot disease of grapevine in Spain, and in Europe. References: (1) S. Alaniz et al. Plant Dis. 91:1187, 2007. (2) S. Alaniz et al. Plant Dis. 95:1028, 2011. (3) E. E. Jones et al. Plant Dis. 96:144, 2012. (4) L. Lombard et al. Mycol. Progress DOI 10.1007/s11557-011-0799-1, 2012.

First Report of Canker Disease Caused by Neofusicoccum australe on Eucalyptus and Pistachio in Spain.

In 2005 and 2006, dieback and branch cankers were observed in 12-year-old Eucalyptus globulus Labill. plantations in Gijón (northern Spain) and a 20-year-old pistachio (Pistacia vera L.) plantation in Constantí (northeastern Spain). Isolations were made from symptomatic branches. Small pieces of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl and plated onto malt extract agar amended with 0.5 g L-1 streptomycin sulfate. Plates were incubated at 25°C in the dark and all growing colonies were transferred to potato dextrose agar (PDA). A Neofusicoccum sp. was consistently isolated from necrotic tissues of both host species. On PDA at 25°C, isolates developed a moderately dense mycelium, initially with a pale yellow pigment diffusing into the medium but becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile eucalyptus and pistachio twigs placed on the surface of water agar after 1 month. Conidia were hyaline, fusiform, aseptate, with granular contents. Conidia from eucalyptus isolates measured (22.5-) 25.4 (-28.1) × (5-) 6.2 (-7.5) µm, (n = 40) and (20.0-) 23.6 (-28.0) × (6.5-) 7.1 (-8.0) µm, (n = 40) from pistachio isolates. Isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (1,2). DNA sequences of the rDNA internal transcribed spacer region (ITS), part of the beta-tubulin (BT2), and part of the translation elongation factor 1-alpha (EF1-α) genes from isolates CBS 122027 (pistachio) and CBS 122026 and CBS 122025 (eucalyptus) were used to confirm the identifications through BLAST searches in GenBank. Representative sequences of all studied regions were deposited in GenBank (ITS: EU375516 and EU375517; BT2: EU375520; EF1-α: EU375518 and EU375519). Pathogenicity tests were conducted on 8-month-old eucalyptus seedlings and 2-year-old pistachio plants with the three N. australe strains mentioned above. A mycelial plug taken from the margin of an actively growing colony of each isolate was put in a shallow wound (0.4 cm2) made with a scalpel on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate and plant species were used, with an equal number of control plants. Plants were maintained in a greenhouse at 25°C. After 3 weeks, all eucalyptus seedlings showed leaf wilting, stem canker, and pycnidia formation around the inoculation site. No foliar symptoms were observed in pistachio plants after 3 months, but depressed cankers variable in size and pycnidia formation developed around the inoculation site. Vascular necroses that developed on the inoculated plants were 10.2 ± 1.2 cm long in eucalyptus and 6.4 ± 1.6 cm long in pistachio, significantly greater than their respective controls (P < 0.01). There were no significant differences in necrosis lengths among the three N. australe isolate inoculations, irrespective of the inoculated host. These results point to a high susceptibility of eucalyptus to N. australe. No symptoms were visible in the control seedlings and no fungus was isolated from them. The pathogen was reisolated from all inoculated plants. To our knowledge, this is the first report of N. australe causing canker disease on eucalyptus and pistachio trees in Spain. References: (1) P. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:1030, 2004.

First Report of Leaf Spot and Twig Blight of Rhododendron spp. Caused by Phytophthora hibernalis in Spain.

During the early spring of 2004, an estimated 20% of containerized nursery stocks of Rhododendron spp. in Asturias (northern Spain) were affected by a foliar disease that has reoccurred annually. Leaf spots were dark brown to almost black, generally oval to round, visible from both sides of the leaf, and expanded to affect the entire leaf including the petiole. Affected leaves abscised from the plant. A Phytophthora sp. was consistently isolated from symptomatic leaf tissues on PARBH medium (3) and hyphal tips were transferred onto potato dextrose agar (PDA). Colonies grown on PDA at 20°C were submerged, had a growth rate of 2.2 mm/day, and had lobes of compact mycelium. Sporangia were semipapillate and caducous with a pedicel (20.0-) 37.7 (-52.5) µm long. Sporangia were asymmetrical in shape with the broadest point near the apex: 25.2 to 40.4 µm long × 10.2 to 15.8 µm wide (average 33.1 × 12.6 µm), and length/width ratio was 2.8:1. Chlamydospores were not observed. Isolates were homothallic and oogonia ranged from 26.5 to 27.5 µm in diameter. Antheridia were mostly amphigynous but occasionally paragynous. Oospores were plerotic and 23.1 to 25.5 µm in diameter. These characteristics conformed to those of Phytophthora hibernalis Carne (2). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. hibernalis (Accession No. AY827556.1 from Citrus sp.). For pathogenicity tests, four isolates of P. hibernalis were used to inoculate detached leaves of Rhododendron hybrid Brigitte. The underside of five detached leaves was inoculated with a drop of 40 µL of a suspension of 104 zoospores/ml. Controls were inoculated with a 40-µL drop of sterile distilled water. Leaves were incubated in a moist chamber at 20°C in the dark. A quantification of the lesion area was made 8 days after inoculation using the software Assess-APS. All inoculated leaves developed necrotic lesions that ranged from 0.246 to 1.512 cm2. P. hibernalis was reisolated from infected tissue. Symptoms were not detected on the controls. The test was repeated twice and similar results were obtained each time. P. hibernalis has been described previously as causing brown rot on citrus in Spain (4) and was isolated from rhododendron plants in California and Oregon (1). To our knowledge, this is the first record of P. hibernalis causing foliar blight on Rhododendron species in Spain as well as in Europe. References: (1) C. Blomquist et al. Online publication. doi:10.1094/PHP-2005-0728- 01-HN. Plant Health Progress, 2005. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (3) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (4) J. J. Tuset. An. Inst. Nac. Investig. Agrar. Ser. Prot. Veg. N.7, 1977.

Outbreak of Pitch Canker Caused by Fusarium circinatum on Pinus spp. in Northern Spain.

During the winter of 2003-2004, dieback symptoms were observed on Pinus radiata and P. pinaster in pine nurseries in Asturias (northern Spain). Small groups of affected seedlings appeared randomly distributed throughout the nurseries. The seedlings died rapidly, showing basal needle dieback, stem lesions, resin exudations, and wilting. Isolations from infected material onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate and Komada's medium consistently yielded Fusarium sp. cultures. The isolates were transferred to PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The cultures were identified as Fusarium circinatum Nirenberg & O'Donnell (= Fusarium subglutinans Wollenweb. & Reinking), causal agent of pitch canker disease, on basis of the presence of polyphialides and characteristic sterile, coiled, hyphae (2). To further confirm their identity, a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) based on histone H3 gene sequences (4) and a test based on the F. circinatum-specific primers, CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment of the intergenic spacer region of the nuclear ribosomal operon (3), were used. Results obtained with both techniques confirmed the morphological identification of the cultures. A representative culture has been placed in the Centraalbureau voor Schimmelcultures (CBS 117843). The pathogen was isolated only from seedlings of P. radiata and P. pinaster. Other species such as P. nigra, P. sylvestris, and Pseudotsuga menziesii, which were also grown in these nurseries, did not show symptoms. Pathogenicity was confirmed by inoculating 6- to 9-month-old P. radiata and P. pinaster seedlings. Small strips of bark (10 × 1 mm) were cut from the stems and similar sized pieces of PDA colonized by F. circinatum were placed in contact with the open wounds and covered with parafilm. Basal needle dieback was observed 10 days after inoculation that resulted in wilting of the seedlings. F. circinatum was reisolated from the affected stems fulfilling Koch's postulates. Later in the year, symptoms of pitch canker were also observed on 20-year-old P. radiata in one forest plantation in Cantabria (northern Spain). Infected branches and shoots of the trees exudated abundant resin, resulting in resinous cankers. The needles, distal to branch tip infections, wilt, fade to yellow then red, and fall from the tree. Affected trees showed noticeable crown dieback. The isolations from the cankers also yielded F. circinatum cultures that were identified as described above. Although a nonrefereed report appeared in 1998 (1), to our knowledge, this is the first report of F. circinatum on P. radiata and P. pinaster in Spain and in Europe. References: (1) L. D. Dwinell et al. Int. Congr. Plant Pathol. 7th. 3:9, 1998. (2) H. I. Nirenberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004. (4) E. T. Steenkamp et al. Appl. Environ. Microbiol. 65:3401, 1999.

Evaluation of the potential causes of epistaxis in dogs with natural visceral leishmaniasis.

Haemostasis was evaluated in 19 dogs with natural Leishmania infection, six of them with a history of epistaxis, and the results were compared with the results from 24 healthy dogs. In addition, the dogs' blood pressure was measured and biopsies were taken from the nasal mucosa. Buccal mucosa bleeding time was prolonged in the dogs with leishmaniasis (P < 0.002) and most significantly in those with epistaxis (P < 0.005). None of the Leishmania-infected dogs had thrombocytopenia, low levels of plasma von Willebrand factor antigen, a prolonged prothrombin time or activated partial thromboplastin time, a low plasma fibrinogen concentration or high serum fibrin degradation products. These results rule out defects of secondary haemostasis or disseminated intravascular coagulation as significant causes of epistaxis in non-complicated leishmaniasis. Histopathology of the nasal mucosa of 10 of the affected dogs, three of them with epistaxis, revealed ulcerative and inflammatory lesions in all of them.

Pathovars of Pseudomonas syringae causing bacterial brown spot and halo blight in phaseolus vulgaris L. are distinguishable by ribotyping.

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported.

Comparison of cyclosporin A and dexamethasone in the treatment of canine nictitans plasmacytic conjunctivitis.

Thirteen dogs with nictitans plasmocytic conjunctivitis were treated with 2.0 per cent cyclosporin drops in the right eye and with 0.1 per cent dexamethasone ointment in the left eye. The response to both therapies was monitored for six weeks, repeat biopsy specimens were taken, and the time for the clinical signs to recur recorded. Conjunctival cultures were taken before and after both therapies. There were no significant differences between the treatments in the remission of clinical signs, the reduction of inflammatory infiltrate in the biopsy specimens, or the time to recurrence of the condition or its subsequent severity. However, the eyes treated with 0.1 per cent dexamethasone tended to recover more rapidly than the eyes treated with 2.0 per cent cyclosporin, and the eyes treated with 2.0 per cent cyclosporin tended to be protected from a recurrence for longer than the eyes treated with 0.1 per cent dexamethasone.

Genetic heterogeneity of clinical strains of Yersinia enterocolitica traced by ribotyping and relationships between ribotypes, serotypes, and biotypes.

A series of 74 Yersinia enterocolitica clinical strains collected in a Spanish region and 10 reference strains, assigned to nine serotypes and five biotypes, were analyzed by ribotyping procedures. Riboprobing, performed separately with HindIII and BglI and using an rrn operon as the probe, generated 13 and 11 ribotypes (discrimination index [DI] = 0.56 and 0.55), respectively. PCR ribotyping, performed with primers complementary to conserved regions of 16S and 23S rRNA genes, generated 13 ribotypes (DI = 0. 56). A combination of data from the three procedures allowed for further discrimination into 17 combined ribotypes (DI = 0.83). The dendrogram obtained by cluster analysis of data from riboprobing indicated a high heterogeneity of the ribosomal DNA regions of the strains under study (similarities between 10 and 92%), which were grouped into three clusters at a similarity level of 0.32. The major cluster included 10 branches, and 7 of these formed a subcluster (similarity coefficient, >83%) represented by strains of serotype O:3 and biotype 2, 3, or 4. The second cluster included four branches, represented by strains belonging to seven non-O:3 serotypes, biotypes 1A and 2, and two of these branches included pyrazinamidase-positive as well as pyrazinamidase-negative strains. The remaining three branches, represented by O:3-biotype 4 strains, formed a third cluster weakly related to the others. Data from this study showed that Y. enterocolitica O:3 organisms assigned to a prevalent and endemic lineage and non-O:3 organisms assigned to three other less-frequent lineages are circulating and causing human disease in the Spanish region under study.

Evaluation of PCR-based methods and ribotyping performed with a mixture of PstI and SphI to differentiate strains of Salmonella serotype Enteritidis.

The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or 'PS ribotyping', was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and SphI, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PT11 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that 'PS ribotyping' was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.

A three-way ribotyping scheme for Salmonella serotype Typhimurium and its usefulness for phylogenetic and epidemiological purposes.

Ribotyping of Salmonella serotype Typhimurium strains was optimised as a tool for epidemiological and phylogenetic purposes. Of five restriction endonucleases evaluated on a series of 84 isolates, HincII, SalI and PvuII were the most useful, generating 13, 9 and 9 ribotypes with 17, 11 and 18 polymorphic restriction sites, and attaining a discrimination index (DI) of 0.81, 0.53 and 0.59, respectively. The combination of results from tests with the three enzymes provided further discrimination (19 ribotypes, DI=0.84). It proved useful for clonal analysis, defining 19 clonal lines with a remarkable degree of genetic heterogeneity, that were grouped into two major clusters (including 12 and 7 lines, respectively) at a significance level of 0.65. When the attributes of this system were compared with those of phage typing, it was found that ribotyping showed higher typability and sensitivity, supporting its use as an appropriate molecular method. In tracing the molecular epidemiology of Typhimurium strains in Asturias, six lines were found that could be considered endemic and were represented by organisms implicated in salmonellosis throughout the period of study; another four lines included organisms isolated from meat, water or both.

Usefulness of phage typing and "two-way ribotyping" to differentiate Salmonella enteritidis strains.

The capacity to differentiate Salmonella enteritidis strains by phage typing and "two-way ribotyping" performed with PstI and SphI was evaluated. The typeability was 96.8% in phage typing and 100% in ribotyping. The series was differentiated into 13 phage types, 19 combined ribotypes, and 39 subtypes or clonal lines by combining results from both methods (of which 11, 13, and 35, respectively, were represented by natural strains). Ribotyping differentiated strains ascribed to PTs 1, 4, 6a, 7, 8, RDNC and UPT. Conversely, some strains of PTs 1, 4, 5a, 6, 6a, 7, 34, RDNC and UPT fall into the most frequent combined ribotype. A dendrogram of genetic similarity generated from the combined ribotypes was traced, and, at a 0.82 similarity level, it showed a major cluster (including 17 combined ribotypes, 88.4% strains ascribed to all PTs tested except PT11), a minor cluster, and four additional lines more loosely related.

Epidemiological differentiation of pathogenic strains of Salmonella enteritidis by ribotyping.

The usefulness of two-way ribotyping, performed with SphI and PstI, as a genetic marker for a series of pathogenic Salmonella enteritidis strains is reported. Eighteen combined ribotypes were differentiated, a discrimination index of 0.77 was reached, a genetic relationship dendrogram was traced, and the results were applied in an epidemiological study.

Discriminatory power and application of ribotyping of Yersinia enterocolitica O:3 in an epidemiological study.

Ribotyping performed with five restriction endonucleases was used in an attempt to subtype Yersinia enterocolitica serotype O:3 and also as a tool for clonal analysis. DNA from organisms under study (48 isolates from diarrheic human feces, 24 from food, and 5 reference strains) was tested by Southern hybridization using a DNA probe carrying an rRNA operon from Escherichia coli. Strains were grouped into seven ribotypes by the HindIII restriction endonuclease, into five ribotypes by Ncil, Bg/l, and Sa/l; and into two ribotypes by EcoRI, resulting in a discrimination index (DI) of 0.37, 0.17, 0.43, 0.13, and 0.03 for the five endonucleases. By combining the results obtained with two or more restriction endonucleases, a further discrimination was registered, the most efficient combination (in terms of discriminatory power vs. cost in work, time, and money) for routine typing being HindIII-Bg/l (9 types, DI=0.58). In the clonal analysis, results obtained with the five restriction endonucleases allowed us to define 11 groupings or clonal lines, which showed a remarkable degree of genetic heterogeneity (genetic distance coefficients between 0.03 and 0.73) and were grouped into two major clusters. One cluster included 93% of the strains and eight lines. At least two of the most frequent lines can be considered endemic in Asturias, Spain, because organisms belonging to these lines have been circulating and causing human yersiniosis in recent years and have also been isolated from commercial raw meat products. Two Ncil ribotypes from the series under study (92.2% of strains included in the prevalent cluster) were similar but not identical to ribotypes of 0.3 organisms from other geographic areas described in the literature, indicating that the genetic structure of prevalent human pathogens of this serotype is basically clonal.

Yersinia enterocolitica O:3. Antimicrobial resistance patterns, virulence profiles and plasmids.

Antimicrobial resistance patterns (ARP), virulence profiles and plasmids in clinical isolates of Yersinia enterocolitica serotype O:3 were studied. The ARP was tested using the disk method. All strains were susceptible to amoxicillin/clavulanic acid, cefoxitin, fosfomycin, gentamicin, kanamycin, neomycin, tetracycline, nalidixic acid, norfloxacin, ciprofloxacin and trimethoprim. All of them presented resistance to ampicillin, all with the exception of one to cephalotin, differing in resistance or susceptibility to chloramphenicol, streptomycin (Sm), sulfadiazine (Sd) and cotrimoxazole. Due to these differences they were grouped into 8 ARP. Twenty-nine strains carried plasmids and were grouped into 5 plasmid profiles. All strains carrying 42 MDa plasmids were positive for virulence tests (calcium dependence, crystal violet binding, Congo red binding and autoagglutination). No correlation between ARP and plasmid profile was found, although small plasmids of 6.5 and 4.1 MDa mediated resistance to Sm-Sd, as was shown by transformation to Escherichia coli strains. DNAs from plasmids were analyzed by restriction enzymes. All 42 MDa plasmids showed identical EcoRI and HindIII profiles. The two 6.5 MDa plasmids showed identical BglII, AvaI and SalI restriction profiles and the four 4.1 Mda plasmids also yielded restriction profiles similar to each other, but different from the 6.5 ones.