Restriction Digest Patterns Research Articles

Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Heterologous Expression and Molecular Cloning from Williamsia Marianensis

The majority of therapy methods include downsides and limits. As a result, many researchers are focused on developing effective remedies. Therapeutic peptides, like proteins and antibodies, are a potential class of medications that have a number of advantages over traditional pharmaceuticals. Williamson marianensis-produced cholesterol oxidase has been demonstrated to have medicinal value. Using PCR and primers specific to an expression vector (pET28b), we were able to clone the cholesterol oxidase gene and express it in E. coli (BL-21/DE3) Rosetta following identification with IPTG. Genscript Corporation in the United States sequenced gyncholestrol oxidase (500 bp) to create a cox sequence, which was then submitted for synthesis. pET 28a(+) cox William showed a twofold restriction digestion pattern. The pattern was made up of two strands: one was a carrier plasmid (4200 bp) and the other was a 2800 base pair strand that contained the cholesterol oxidase gene. The cholesterol oxidase gene was successfully cloned and expressed as a consequence. Williamson marianensis-derived cholesterol oxidase will be exploited in future medicinal re

Food-borne outbreak of Listeria monocytogenes in school students in Seoul, Korea

In September 2018, an outbreak of Listeriosis cases in Korea was traced to food involved, using retrospective cohort studies and PFGE analyses. This study aimed to describe the first L.monocytogenes outbreak identified in Korea. We confirmed the presence of Serovar (4C) and virulence genes, and evaluated the genetic correlation between isolates by restriction digestion patterns of ApaI and AscI. Based on the epidemiological association, it is presumed that the seasoned crab meat with bean sprouts are contaminated by cross contamination during the bean sprouts washing (relative risk was 1.24; p-value: 0.0021 and they possessed virulence genes. Therefore, active laboratory surveillance is necessary to recognize the risk of L. monocytogenes in Korea.

Comparison of single and multi-host enrichment approach for harnessing lytic phages against antimicrobial-resistant E. coli: Repurposing the enrichment step

Escherichia coli has been enlisted as a pathogen of antimicrobial resistance (AMR) importance posing a threat to human health. In the wake of the widespread AMR, lytic bacteriophages remain a promising alternative to antibiotics for controlling the multidrug-resistant (MDR) pathogens. However, the major hindrance for phage therapy is the lack of a rapid procedure to harness broad host range phages. Hitherto in vogue, lytic phage isolation methods are based on conventional single host enrichment (M1). Limitation to the available methods includes a higher number of steps, additional instrumentation, cost and time involved in the screening of samples and more recovery of narrow host range phages.To improve the conventional method of isolation, a multiple host enrichment approach (modified method; M2) is developed and validated with a classical approach using 24 MDR extended-spectrum β-lactamase producing (ESBL) E. coli and universal coliphage host E. coli NCIM 2089. A total of 58 phages were isolated using both the methods (27 phages from M1 and 31 phages from M2) and were categorized, based on the DNA restriction digestion pattern, to 25 groups with a maximum host range of 17 in host range analysis. Even though there is no significant difference (P > 0.01) in recovering number of phages between M1 and M2 methods, excepting in one occasion where MFB13 host yielded 3 more morphologically distinct phages in M2 (p ≤ 0.05), the M2 approach was better in terms of reduction in time (4.72%) and cost (23.5%) and same instrumentation requirement. This emphasizes that the proposed method has the potential to yield broad host range lytic phages against localized strain repository of AMR E. coli which can be leading to its application in food, food contact surfaces as well as phage therapy.

Epidemiological Study of an Outbreak of KPC-2-producing Klebsiella pneumoniae in a Tertiary Hospital in Korea

Background: The prevalence of carbapenemase-producing Enterobacteriaceae (CPE), especially the KPC-2-producing Klebisella pneumoniae, is rapidly increasing and becoming a menace to global public health. This study aims to present the molecular epidemiology of the KPC-2-producing K. pneumoniae isolates emerged in a tertiary hospital in South Korea and describe its clinical significance. Methods: This study included carbapenem-resistant K. pneumoniae isolates collected from a tertiary hospital from April to December in 2018. Antimicrobial susceptibility of K. pneumoniae isolates was tested using disk diffusion method. PCR and DNA sequence analyses were performed to identify the resistance genotype. In addition, the molecular epidemiology was investigated using pulsed-field gel electrophoresis (PFGE) and multilocus sequencing typing (MLST). Results: Total 100 KPC-2-producing K. pneumoniae isolates were collected, which were mainly classified into two pulsotypes according to the XbaI restriction digestion pattern by PFGE analysis (pulsotype A, n = 31; pulsotype B, n = 63). The isolates exhibiting pulsotype A belonged to ST395 and the remaining isolates exhibiting pulsotype B were attributed to ST307 by MLST analysis. Conclusion: This study investigated clinical information and molecular bacterial profiles for KPC-2-producing K. pneumoniae isolates. These findings indicate that the proper infection control activities are needed to prevent the spread of multidrug-resistant organisms such as CPE, which could cause high mortality in clinical field.

Green approach to phytopathogen: Characterization of lytic bacteriophages of Pseudomonas sp., an etiology of the bacterial blight of pomegranate

Two morphologically different bacteriophages were isolated from the river and soil samples from various locations of Maharashtra, India against the phytopathogen Pseudomonas sp. that was recently reported to cause a new bacterial blight of pomegranate. Both the phages belonged to the order Caudovirales representing the families Siphoviridae (vB_Psp.S_PRɸL2) and Myoviridae (vB_Psp.M_SSɸL8). The multiplicity of infection ranged from 0.01 to 0.1, phage adsorption rate from 39% to 66%, latent period from 10 to 20 min with a burst size of 24–85 phage particles per infected host cell. The genome size of phages PRɸL2 and SSɸL8 was approximately 25.403 kb and 29.877 kb respectively. Restriction digestion pattern of phage genomic DNA was carried out for phage PRɸL2, Eco RI resulted in two bands and Hind III resulted in three bands while for phage SSɸL8, both Eco RI and Hind III each resulted in three bands. SDS-PAGE protein profile showed six bands for PRɸL2 and nine bands for SSɸL8 of different proteins. Phages showed high pH stability over a range of 4–9, temperature stability over a range of 4–50 °C and UV radiation showed a reduction up to 89.36% for PRɸL2 and 96% for SSɸL8. In short, the present research work discusses for the first time in-detailed characterization of phages of a phytopathogen Pseudomonas sp. from Maharashtra, India, which can be further efficiently used for biological control of the causative agent of a new bacterial blight disease of pomegranate.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

Prevalence, Serotype Diversity, Genotype and Antibiotic Resistance of Listeria monocytogenes Isolated from Carcasses and Human in Korea.

This study investigated the prevalence of Listeria monocytogenes in slaughterhouses, and determined serovars and genotypes, and antibiotic resistance of the isolates obtained from slaughterhouses and humans in Korea. Two hundred ninety samples were collected from feces (n=136), carcasses [n=140 (cattle: n=61, swine: n=79)], and washing water (n=14) in nine slaughterhouses. Eleven human isolates were obtained from hospitals and the Korea Center for Disease Control and Prevention. Listeria monocytogenes was enriched and identified, using polymerase chain reaction (PCR) and 16S rRNA sequencing. Serovars and presence of virulence genes were determined, and genetic correlations among the isolates were evaluated by the restriction digest patterns of AscI. Antibiotic resistance of L. monocytogenes isolates were examined against 12 different antibiotics. Of 290 slaughterhouse samples, 15 (5.17%) carcass samples were L. monocytogenes positive. Most L. monocytogenes isolates possessed all the virulence genes, while polymorphisms in the actA gene were found between carcass and human isolates. Serovars 1/2a (33.3%) and 1/2b (46.7%) were the most frequent in carcass isolates. Genetic correlations among the isolates from carcass and clinical isolates were grouped within serotypes, but there were low geographical correlations. Most L. monocytogenes isolates were antibiotic resistant, and some strains showed resistance to more than four antibiotics. These results indicate that L. monocytogenes are isolated from carcass and human in Korea, and they showed high risk serotypes and antibiotic resistance. Therefore, intensive attentions are necessary to be aware for the risk of L. monocytogenes in Korea.

Acinetobacter baumannii transfers the blaNDM-1 gene via outer membrane vesicles.

To investigate the transmission of the gene encoding New Delhi metallo-β-lactamase-1 ( bla NDM-1 ) through outer membrane vesicles (OMVs) released from an Acinetobacter baumannii strain (A_115). Isolation and purification of OMVs by density gradient from a carbapenem-resistant clinical strain of A. baumannii harbouring plasmid-mediated bla NDM-1 and aac(6')-Ib-cr genes was performed. DNA was purified from the OMVs and used for PCR and dot-blot analysis. Vesicles treated with DNase I and proteinase K were used to transform A. baumannii ATCC 19606 and Escherichia coli JM109 strains. MIC values for the transformants were determined, followed by PCR and restriction digestion of plasmids. PFGE was done for A_115 and transformants of ATCC 19606 and JM109. The A. baumannii strain (ST 1462) released vesicles (25-100 nm) during in vitro growth at late log phase. PCR and dot-blot analysis confirmed the presence of bla NDM-1 and aac(6')-Ib-cr genes in intravesicular DNA. bla NDM-1 and aac(6')-Ib-cr genes were transferred to both the A. baumannii ATCC 19606 and E. coli JM109 recipient cells. The transformation frequency of the purified OMVs was in the range of 10 -5 -10 -6 and gradually reduced with storage of OMVs. The sizes of the plasmids in the transformants and their restriction digestion patterns were identical to the plasmid in A_115. The transformants showed elevated MIC values of the β-lactam group of antibiotics, which confirmed the presence of a bla NDM-1 -harbouring plasmid. This is the first experimental evidence of intra- and inter-species transfer of a plasmid harbouring a bla NDM-1 gene in A. baumannii via OMVs with high transformation frequency.

Prevalence and Genetic Characteristics of Meatborne Listeria monocytogenes Isolates from Livestock Farms in Korea

This study aimed to evaluate the prevalence of Listeria monocytogenes on livestock farms in Korea and determine their serotypes and genetic correlations. Twenty-five livestock farms in Korea (central: 15, south west: 7, south east: 3) were visited 2-3 times, and 2,018 samples (feces: 677, soil: 680, silage: 647, sludge: 14) were collected. Samples were enriched in LEB (Listeria enrichment broth) and Fraser broth media, and then plated on Palcam agar. The isolates were identified by PCR and 16S rRNA gene sequencing. Then, the serotypes, presence of virulence genes (actA, inlA, inlB, plcB, and hlyA), and antibiotic resistance were determined. Genetic correlations among the isolates were evaluated by analyzing the restriction digest pattern with AscI. Of the 2,018 samples, only 3 (0.15%) soil samples (FI-1-FI-3) from 1 farm in the south east region were positive for L. monocytogenes. Based on biochemical tests and multiplex PCR, the serotype of the isolates were 4ab (FI-1 and FI-3) and 3a (FI-2), which are not common in foodborne L. monocytogenes. The 3a serotype isolate was positive for all tested virulence genes, whereas the 4ab serotype isolates were only positive for hlyA, actA, and inlA. The isolates were resistant to all 12 tested antibiotics, especially FI-3. The genetic correlations among the isolates were 100% for those of the same serotype and 26.3% for those of different serotypes. These results indicate that the prevalence of L. monocytogenes on livestock farms in Korea is low; however, the isolates are pathogenic and antibiotic resistant.

Production of bioactive protein hydrolysate using the yeasts isolated from soft chhurpi

The aim of this work was to study the production of bioactive protein hydrolysates using yeasts isolated from chhurpi. For this, a total of 125 proteolytic yeasts were isolated and molecular identification was carried out by analysis of the restriction digestion pattern generated by digesting the PCR amplified internal transcribed spacer region and 5.8S rRNA gene (ITS1-5.8S-ITS2) using three endonucleases (HaeIII, CfoI and HinfI). The results obtained showed that different proteolytic yeasts were dominant in marketed products (Kluyveromyces marxianus and Issatchenkia orientalis) and samples from production centers (Trichosporon asahii, Saccharomyces cerevisiae and Exophiala dermatitidis). Proteolytic strains in individual groups showed their ability to hydrolyze milk protein and enhance antioxidant property. Among the isolates, fermentation using K. marxianus YMP45 and S. cerevisiae YAM14 resulted in higher antioxidant activity. This is the first report on application of yeast isolated from fermented food of North-East India for the production of bioactive protein hydrolysate.

MboII Restriction Fragment Length Analysis Of Estrogen Receptor 1 Gene

IntroductionCervical cancer is the commonest genital tract malignant neoplasm that affect women worldwide. It is most notable in the lower‐resource countries of sub‐Saharan Africa. Understanding the restriction digestion pattern of Human estrogen receptor‐α gene (ESR‐1) would provide information on the possible mutations on the gene that can serve as potential diagnostic and therapeutic markers.The present work is focused on determining the MboII restriction fragment length on esr1 gene of cervical cancer cells. The elucidation of this is vital in the diagnosis of the cervical cancer diseaseMaterials and MethodPCR technique was used to amplify the promoter region of exon1 of the esr1 gene for ten samples. MboII restriction enzyme specific for the promoter site was used to carry out restriction fragment analysis.Result and DiscussionThe result indicates that the ESR1 gene for cervical cancer has restriction patterns or sequences recognized by the restriction enzyme. It is important to note however, that there is need for similar work using large sample size in order to conclusively ascertain the presence of polymorphism in the ESR 1 promoter region and the restriction fragment length of the ESR1 gene. Seven out of the ten samples analyzed for restriction fragment length exhibited the presence of restriction site specific to the MboII restriction endonuclease. These restriction sites are vital for diagnostic purposes.ConclusionThe result indicates that the ESR1 gene for cervical cancer has restriction patterns or sequences recognized by the restriction enzyme.Support or Funding InformationNo conflict of Interest Disclosure

Growth inhibitory effect of bacteriophages isolated from western and southern coastal areas of Korea against Vibrio parahaemolyticus in Manila clams

Vibrio parahaemolyticus is one of the leading causes of seafood-related illnesses in Korea and the whole world. The growth inhibitory effect of V. parahaemolyticus-specific phages (Vpp) in clam samples was investigated. Six bacteriophages specific to V. parahaemolyticus were isolated by the agar overlay method using sediment and seafood samples from the western and southern coastal areas of Korea. Host range, restriction digestion pattern, and transmission electron microscope images of phage isolates were examined. The most effective phage, Vpp2, at a multiplicity of infection (MOI) of 10 showed 4.2 log CFU/mL reduction in V. parahaemolyticus growth at 30 °C and 6 log CFU/mL reduction after the incubation at 37 °C for 6 h. In a food application study, Vpp2 at an MOI of 100 demonstrated a 2.1 log CFU/mL reduction at 25 °C after a 24-h incubation in shell-less Manila clams spiked with V. parahaemolyticus. Additional studies are needed to characterize the newly discovered Vpp and their potential applications as biocontrol agents in food.

Inquiry-based experiments for large-scale introduction to PCR and restriction enzyme digests.

Polymerase chain reaction and restriction endonuclease digest are important techniques that should be included in all Biochemistry and Molecular Biology laboratory curriculums. These techniques are frequently taught at an advanced level, requiring many hours of student and faculty time. Here we present two inquiry-based experiments that are designed for introductory laboratory courses and combine both techniques. In both approaches, students must determine the identity of an unknown DNA sequence, either a gene sequence or a primer sequence, based on a combination of PCR product size and restriction digest pattern. The experimental design is flexible, and can be adapted based on available instructor preparation time and resources, and both approaches can accommodate large numbers of students. We implemented these experiments in our courses with a combined total of 584 students and have an 85% success rate. Overall, students demonstrated an increase in their understanding of the experimental topics, ability to interpret the resulting data, and proficiency in general laboratory skills.

Prevalence and genetic profiles of Escherichia coli from mangroves and mangrove associated foods off Goa, India

A total of 120 samples comprising of water (45), sediment (45) and mangrove originated food (30) collected from mangrove ecosystems of Goa were screened for Escherichia coli employing ISO-16654 method. Seventy-one (59.16%) samples were positive for E. coli. The E. coli isolates were further characterized by serotyping, virulence gene profiling and pulsed field gel electrophoresis (PFGE). Water and sediment samples were analyzed for physico-chemical parameters. The serotypes reported were O1, O10, O13, O17, O36, O41, O50, O68, O105, O116, O141, O148, O159, O162 and rough types while, 23 strains could not be typed. The stx1 and stx2 genes were detected in 33(46.47%) and 16(22.53%) isolates, respectively. The XbaI restriction digestion patterns of the stx positive strains were diverse. Interestingly, few strains isolated from diarrheal patients and from water, sediment and food from mangrove sources were genetically similar. The study showed that the mangrove ecosystem could be a potential reservoir for pathogenic E. coli.

Molecular characterization of lactobacilli isolated from fermented idli batter

Lactic acid bacteria are non pathogenic organism widely distributed in nature typically involved in a large number of spontaneous food fermentation. The purpose of this study was to characterize the bacteriocinogenic lactobacilli from fermented idli batter which can find application in biopreservation and biomedicine. Eight most promising lactobacilli were chosen from twenty two isolates based on their spectrum of activity against other lactic acid bacteria and pathogens. The eight lactobacilli were characterized based on the various classical phenotypic tests, physiological tests and biochemical tests including various carbohydrate utilization profiles. All isolates were homo fermentative, catalase, and gelatin negative. Molecular characterization was performed by RAPD, 16S rRNA analysis, 16S ARDRA, and Multiplex PCR for species identification. RAPD was carried out using the primer R2 and M13. Five different clusters were obtained based on RAPD indicating strain level variation. 16S rRNA analysis showed 99 to 100% homology towards Lactobacillus plantarum. The restriction digestion pattern was similar for all the isolates with the restriction enzyme AluI. The subspecies were identified by performing Multiplex PCR using species specific primer. Among the five clusters, three clusters were clearly identified as Lactobacillus plantarum subsp. plantarum, Lactobacillus pentosus, and Lactobacillus plantarum subsp. argentoratensis.

Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms

Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host–bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component.

Tracking the sources of salmonella in ground beef produced from nonfed cattle.

The objective of this study was to determine the source(s) of Salmonella contamination in ground beef. One hundred dairy cows were harvested in a U.S. commercial beef processing plant. Samples of hides, carcasses after hide removal and before exposure to antimicrobial intervention, carcasses after all antimicrobial interventions, superficial cervical lymph nodes from the chuck, trim, ground beef, and air were obtained. Ninety-six percent of the hide samples, 47% of the carcasses before intervention, 18% of the lymph nodes, 7.14% of the trim, and 1.67% of the ground beef samples were positive for Salmonella. None of the samples obtained from the carcasses after the full complement of interventions and none of the air samples were positive for Salmonella. All Salmonella-positive samples were subjected to pulsed-field gel electrophoresis, and eight DNA Xba I restriction patterns were identified. The majority of isolates had one of two restriction digest patterns. The strain isolated from ground beef had the same pattern as the strains isolated from hides and from carcasses immediately after hide removal. The Salmonella isolates from trim samples and lymph nodes also had the same restriction digest pattern. These results indicate that hide and lymph nodes are the most likely sources of Salmonella in ground beef. Dressing practices that effectively reduce or eliminate the transfer of bacteria from hide to carcass and elimination of lymph nodes as a component of raw ground beef should be considered as measures to reduce Salmonella contamination of ground beef. Because total elimination of lymph nodes from ground beef is not possible, other approaches should be explored. Easily accessible lymph nodes could be screened for Salmonella very early in the slaughter process. When the results are positive for Salmonella, the corresponding carcasses should be fabricated separately at the end of the production run, and the trim from these carcasses should be subjected to a treatment that destroys Salmonella.

Comparative genomic analysis of Atlantic salmon, Salmo salar, from Europe and North America

BackgroundSeveral lines of evidence including allozyme analysis, restriction digest patterns and sequencing of mtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon (Salmo salar) from North America and Europe are genetically distinct. These observations are supported by karyotype analysis, which revealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29 pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this map with the well developed map for European Atlantic salmon.ResultsWe used microsatellite markers, which had previously been mapped in the two Atlantic salmon SALMAP mapping families from the River Tay, Scotland, to carry out linkage analysis in an Atlantic salmon family (NB1) whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences in recombination rates between female and male Atlantic salmon have been noted, separate genetic maps were constructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male map has 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27 composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from the Western Atlantic.ConclusionsA comparison of the composite NB1 and SALMAP linkage maps revealed the reason for the difference in the chromosome numbers between European and North American Atlantic salmon: Linkage groups AS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into a single NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it will require finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison of the genomic architecture of Atlantic salmon from North America and Europe with respect to chromosome organization.

Plasmid Profiles and Prevalence of Intermediately VirulentRhodococcus equifrom Pigs in Nakhonpathom Province, Thailand: Identification of a New Variant of the 70-kb Virulence Plasmid, Type 18

The prevalence of intermediately virulent Rhodococcus equi isolates from pig submaxillary lymph nodes from four slaughterhouses in Nakhonpathom province, Thailand, was investigated. The isolates were tested for the presence of virulence plasmids and the 20-kDa virulence-associated protein antigen (VapB) gene by PCR. Of the 734 submaxillary lymph nodes tested, 19 (2.6%) produced positive cultures of R. equi. All 19 isolates were positive for the VapB gene and contained virulence plasmids that were identified as type 1 (six isolates), type 6 (two isolates), type 7 (one isolate), type 16 (two isolates), and a new variant (eight isolates). Based on the restriction digestion patterns of the plasmid DNAs, we tentatively designated the variant as type 18. Investigation of the prevalence and plasmid profiles of VapB-positive R. equi in pigs should be extended throughout Thailand to evaluate potential sources of zoonotic infections.