Restriction Endonuclease Fragment Patterns Research Articles

Isolation and characterization of plaque-purified strains of Malacosoma disstria Nucleopolyhedrovirus

Seven plaque-purified genotypic variants or strains, derived from a previously described field isolate of the Malacosoma disstria Nucleopolyhedrovirus (MadiNPV) from Alberta populations of forest tent caterpillar, were characterized based on distinctive restriction endonuclease fragment patterns. Two strains, MadiNPV-pp3 and MadiNPV-pp11, were selected for further characterization, as they represented strains producing high and low budded virus (BV) titres, respectively, in the M. disstria cell line UA-Md203. Analysis of restriction endonuclease fragment profiles indicated the genomes differed significantly in size, 133.8 +/- 2.4 kb for MadiNPV-pp3 and 118.1 +/- 3.5 kb for MadiNPV-pp11. These strains were characterized based on their BV production in three different cell lines derived from M. disstria haemocytes. Compared with MadiNPV-pp11, MadiNPV-pp3 produced two- to three-fold more BVs in UA-Md203 and 210 other cell lines; however, BV production was only marginally higher for MadiNPV-pp3 in the UA-Md221 cell line. Similarly, the yield of polyhedral inclusion bodies was significantly higher for MadiNPV-pp3 in UA-Md203 and 210 cell lines than for MadiNPV-pp11 but not in the UA-Md221 cell line. This data, although derived from a limited number of cell lines, suggested MadiNPV-pp3 may have a broader tissue tropism than MadiNPV-pp11.

Molecular analysis of methicillin-resistant Staphylococcus aureus reveals an absence of plasmid DNA in multidrug-resistant isolates

The number, diversity and restriction enzyme fragmentation patterns of plasmids harboured by 44 multidrug-resistant hospital-acquired methicillin-resistant Staphylococcus aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK) were compared. In addition, pulsed-field gel electrophoresis (PFGE) type following SmaI chromosomal digest and SCC mec element type assignment were ascertained for each isolate. All MR-HA-MRSA and MR-CA-MRSA isolates possessed the type II SCC mec, harboured no plasmid DNA and belonged to one of five PFGE types. Forty-three out of 50 HA-MRSA isolates and all 34 CA-MRSA isolates possessed the type IV SCC mec and all but 10 of the type IV HA-MRSA isolates and nine CA-MRSA isolates carried one or two plasmids. The 19 non-multidrug-resistant isolates (NMR) that did not harbour plasmids were only resistant to methicillin whereas all the NMR isolates harbouring at least one plasmid were resistant to at least one additional antibiotic. We conclude that although plasmid carriage plays an important role in antibiotic resistance, especially in NMR-HA-MRSA and CA-MRSA, the multidrug resistance phenotype from HA-MRSA is not associated with increased plasmid carriage and indeed is characterised by an absence of plasmid DNA.

Influence of LDL receptor gene polymorphisms on serum lipid levels and response to atorvastatin in subjects with familial and non familial hypercholesterolemia

Mitochondrial DNA codes for enzymes involved in the cellular energetic pathway. The polymorphism of this genome has been extensively analyzed for disease associations, but can also be used to characterize anthropological distances between populations. This study presents the results of mitochondrial DNA (mtDNA) sequence variation for a population sample of 50 unrelated individuals originating from western Algeria. The samples were studied with the recently developed long PCR technique followed by RFLP analysis using six restriction endonucleases: HpaI, BamHI, HaeII, MspI, AvaII and HincII One new morph for HpaI (named Hpal-9Alg) was detected, and was found to be derived from the combination of the already known morphs 3 and 4. mtDNA restriction endonuclease fragment patterns were analyzed for potential site gain or loss and classified into 18 mtDNA types by the sequence-comparison method. Three mtDNA types (97Alg; 2-1-7-1-1, 98Alg; 2-1-1-8-37 and 99Alg; 9Alg-1-1-1-3) were detected for the first time. Another mtDNA marker — the presence of the 9 by deletion in the COII/tRNALys region - was also studied in the Algerian sample. No deletions were observed. Our results indicate that the Algerians are genetically related to the Israeli-Arab population, with certain characteristics found in southern Europeans and others found in sub-Saharan Africans.

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC).

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.

Bovine Staphylococcus aureus mastitis: strain recognition and dynamics of infection.

Restriction enzyme fragmentation pattern (REFP) analysis was used to recognise Staphylococcus aureus strain variation in naturally occurring bovine subclinical mastitis. Multiple colony REFP analysis identified eight distinct strains of S. aureus in addition to the original strains A and B that were infused via the intramammary route, indicating that individual quarters of the udder may be colonised simultaneously by more than one strain of S. aureus. Examination of multiple colonies per milk sample may benefit bacterial strain recognition as an epidemiological tool in mastitis investigations. The dynamics of intramammary infection were determined using a novel double crossover experimental challenge. Quarters remained persistently infected for several weeks following challenge in all four cows, irrespective of the challenge strain. This indicated that no alteration of the original subclinical infection, including the possible induction of clearance of the quarters infected with S. aureus, or replacement of the original strain by the infused strain was induced by challenge. The persistent subclinical infection in all four animals supports previous reports on the chronicity of S. aureus intramammary infection in dairy cows.

Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis

Three hundred and forty-two Streptococcus uberis isolates were cultured from milk samples from subclinical and clinical cases of dairy cattle mastitis. The samples were collected from 15 different New Zealand farming regions, including eight specific farms, during field research trials and veterinary diagnostic investigations. Pulsed-field gel electrophoresis was used to determine and compare the degree of genetic dissimilarity between the restriction endonuclease fragment pattern of the 342 New Zealand and a single United States S. uberis isolate. The 343 isolates exhibited 330 different restriction endonuclease fragment patterns. The United States isolate had a pattern unlike any of the New Zealand isolates. Most of the isolates were genetically different strains (pattern deferred by at least 33%), but identical patterns were noted within the same or different quarters of an individual cow, different cows within the same farm, and from different cows from the same or different districts, farming regions or islands. Seven of the eight selected farms had at most only one pair of isolates with banding patterns, which differed by less than 33%. A high degree of dissimilarity was noted in individual herds in which all the samples were collected on the same day or over a 2-year period. The high degree of dissimilar isolates is an indication that S. uberis infections in New Zealand dairy cattle are largely due to the opportunistic nature of the organism in the cows’ environment. Prevention and treatment of S. uberis mastitis will therefore need to be directed at a multitude of different strains present throughout the country as well as in individual herds.

Mitochondrial DNA polymorphisms of a west Algerian population (Oran region)

Mitochondrial DNA codes for enzymes involved in the cellular energetic pathway. The polymorphism of this genome has been extensively analyzed for disease associations, but can also be used to characterize anthropological distances between populations. This study presents the results of mitochondrial DNA (mtDNA) sequence variation for a population sample of 50 unrelated individuals originating from western Algeria. The samples were studied with the recently developed long PCR technique followed by RFLP analysis using six restriction endonucleases: HpaI, BamHI, HaeII, MspI, AvaII and HincII One new morph for HpaI (named Hpal-9 Alg) was detected, and was found to be derived from the combination of the already known morphs 3 and 4. mtDNA restriction endonuclease fragment patterns were analyzed for potential site gain or loss and classified into 18 mtDNA types by the sequence-comparison method. Three mtDNA types (97 Alg; 2-1-7-1-1, 98 Alg; 2-1-1-8-37 and 99 Alg; 9 Alg-1-1-1-3) were detected for the first time. Another mtDNA marker — the presence of the 9 by deletion in the COII/tRNA Lys region - was also studied in the Algerian sample. No deletions were observed. Our results indicate that the Algerians are genetically related to the Israeli-Arab population, with certain characteristics found in southern Europeans and others found in sub-Saharan Africans.

Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis.

Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees (Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations (AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.

Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis

Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees ( Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations ( AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.

Conservation Genetics of the Common Snapping Turtle (Chelydra serpentina)

Previous studies of relationships among the subspecies of snapping turtles (Chelydra serpentina} based on morphological and osteological characters have been inconclusive. We investigated relationships among the four currently recognized subspecies using restriction endonuclease fragment patterns of mtDNA and protein electrophoresis. Sixteen six‐based recognizing restriction endonucleases yielded 90 variable fragments that define 11 different haplotypes. Individuals of the two North American subspecies, C. s. osceola and C. s. serpentina, are closely related, differing by a maximum of 0.5% sequence divergence. The Central American subspecies, C. s. rossignonii and C. s. acutirostris, are more distinct, both from each other (a minimum of 1.7% sequence divergence) and from the North American samples (an average of 4.45% sequence divergence). The degree of allozymic variation among the four subspecies was found to be limited and could not be used to diagnose the four recognized subspecies. The mtDNA data presented here support the species‐level distinctness of C. s. rossignonii and C. s. acutirostris from each other and from a C. s serpentina‐C. s. osceola complex. The recognition of three distinctive groups of Chelydra rather than one widespread polytypic species has important conservation implications because it focuses attention on the poorly known middle and South American species.

Relationships between populations of Pseudomonas syringae pv. persicae determined by restriction fragment analysis

Pseudomonas syringae pv. persicae, the pathogen causing bacterial decline of stone‐fruit, was first noted almost simultaneously in France and in New Zealand, together with a closely related pathogen from myrobalan plum in England. The relative similarity of 31 strains from these three countries was examined by comparing DNA restriction endonuclease fragment patterns. Fragment patterns produced from digested DNA samples which were electrophoresed on polyacrylamide gels and visualized by silver‐staining, were analysed using the software package Gelcompar. The fragment patterns produced by strains from France and England formed homogeneous but separate groups, while those from New Zealand were relatively heterogeneous. This finding suggests that the New Zealand population of P.s. persicae is older than those found in Europe. Problems in explaining the distribution of the pathogen are discussed.

Molecular analysis of Salmonella enterica serotype Dublin: building bridges between population genetic and molecular epidemiological studies.

Population genetic studies of Salmonella enterica serotype Dublin using multilocus enzyme electrophoresis have recognised two dominant clones termed Du1 and Du3. The characterisation of plasmids in Dublin suggests greater strain diversity. The application of restriction enzyme fragmentation pattern (REFP) analysis of genomic DNA using Sau3A and HincII together with plasmid subtractive analysis can resolve anomalies in earlier comparisons. Twenty-six isolates were selected for inclusion in the study. All had been previously characterised with respect to their plasmids, and were isolated from the USA, Canada and five European countries. On the basis of plasmid profiles, 17 were predicted to correspond with Du1 and Du3. Sau3A digestion generated two distinct REFPs (A and B) of < 70% similarity, which corresponded with Du1 and Du3. After the contribution of plasmid-derived bands was subtracted, two variants of A (A1 and A2) and four of B (B1, B2, B3 and B4) were recognised. Seventeen were concordant with predictions from population genetic studies. Nine that could not be predicted on the basis of atypical plasmid profiles all showed REFP A1 (Du1) and were consistent with the incursion of additional plasmids or plasmid cointegration. REFPs from HincII digests generally corroborated Sau3A data but showed greater overall similarity between the strains and more influence from plasmid DNA.

Diversity and molecular variation among plasmids in Salmonella enterica serotype Dublin based on restriction enzyme fragmentation pattern analysis.

Molecular variation within and between plasmids of Salmonella enterica serotype Dublin was analysed. Such variation has been demonstrated in the serotype-specific plasmids (SSP's) of Typhimurium and Enteritidis. The two aims of this study were to determine the plasmid diversity in a host-adapted serotype and also the incidence of molecular variation in the SSP among strains of Dublin using restriction endonuclease fragmentation pattern (REFP) analysis with Pst1, Sma1 and EcoRV. Sixty-five strains were examined from seven countries. Plasmid profile and REFP analysis showed that none of the strains was plasmid-free. Seventy-seven percent of the strains possessed the 72 kb SSP either alone or in combination with another plasmid; 23% harboured plasmids which were molecular variants of the SSP. Four of the variants were more closely related to each other than to the reference SSP and were harboured by Dublin isolated from both the USA and Europe. A further three were shown to be cointegrate plasmids and were similarly distributed. Thirty-two percent of strains possessed the SSP alone. None of the UK strains was resistant to any of the antimicrobial agents tested whereas 74% of the remaining strains were resistant to between one and five antimicrobial agents. This study corroborates previous findings concerning the high degree of stability of the SSP and confirmed the clonal nature of Dublin. Co-resident plasmids provided evidence of sub-clones within localized geographical areas.

Leishmania (Viannia) lainsoni occupies a unique niche within the subgenus Viannia

Leishmania lainsoni has recently been recognized as a new peripylarian species belonging to the subgenus Viannia and the L. braziliensis complex. It has been isolated from its sandfly vector, reservoir host and cutaneous lesions of human patients. Microscopical examination has shown characteristics which are different from those of other members of the L. braziliensis complex. Nuclear deoxyribnucleic acid (DNA) hybridization patterns with a β tubulin probe and kinetoplast DNA buoyant density measurements show close similarities with other species of the L. braziliensis complex. However, kinetoplast DNA restriction enzyme fragment patterns of L. (V.) lainsoni isolates show similarities to L. mexicana complex species as well as weak cross hybridization. L. (V.) lainsoni is also amplified with L. braziliensis complex specific polymerase chain reaction (PCR) primers but it requires a lower annealing temperature and gives a 300 base pair PCR product. A possible model for the binding of PCR primers to the L. (V.) lainsoni kinetoplast DNA minicircle is proposed.

The distribution of serotype-specific plasmids among different subgroups of strains ofSalmonella entericaserotype Enteritidis: characterization of molecular variants by restriction enzyme fragmentation patterns

Four hundred and thirty-four isolates of Salmonella enterica serotype Enteritidis were studied. They were grouped into five subsets defined by either the collection criteria or the parameter which formed the basis for subsequent analysis. Seventy-seven per cent harboured the serotype-specific plasmid (SSP). In 55% of the isolates this was the sole plasmid. Molecular variation in the SSP was detected in 17 (5%) of the isolates on the basis of restriction enzyme fragmentation pattern (REFP) analysis using Pst I and Sma I. The SSP variants were further characterized using additional restriction enzymes chosen to optimize the information content and analysed using a coefficient of similarity. A variant SSP designated pOG690 showed greater resemblance to the SSP of Salmonella enterica serotype Typhimurium than Enteritidis; 89% and 68% respectively for Pst I and 79% and 55% respectively for Sma I. In respect of the Pst I data pOG690 shared at least 55 kb of DNA with the Typhimurium SSP and 37 kb with the SSP of Enteritidis. This variant was associated with poultry (duck, goose, chicken) and all isolates belonged to phage type 9b. Other variants were associated with phage types 4, 6, 6a, 9a, 11, 15 and 24. The epidemiological implications of these results are discussed.

DPB1 locus PCR-RFLP typing of the fourth Asia-Oceania Histocompatibility Workshop cell panel reveals a novel DPB1 allele.

DPB1 locus typing of the 155 cell 4AOHW panel was performed using a PCR-RFLP method. Ambiguity of allele assignment was resolved by amplification using sequence-specific primers. Of the 150 cells for which typings were achieved, three exhibited unusual restriction enzyme fragment patterns, suggesting the possibility of novel DPB1 alleles. Sequence analysis revealed one allele present in the currently reported 46, one novel allele (4AOHW/107) not present among the 46, and one from a non-human primate which is being investigated. Twenty-six (26) of the 34 10IHW cells have been studied previously by cDNA RFLP, and strong haplotypic associations have been demonstrated between DPA1 and DPB1 locus alleles. It is proposed that exploitation of intron polymorphisms making haplotypes will be an integral part of future DPB1 typing as a "first-pass' stratification process to minimize the requirement for sequence-based methods to definitively assign DPB1 alleles.

Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae

To assess the role of capsular serotypes in the virulence of Streptococcus pneumoniae, we have constructed isogenic derivatives differing only in the type of capsule expressed. Strains of types 2, 5, and 6B were converted to type 3 by transformation and selection for an erythromycin resistance marker linked to the type 3 capsule locus. Characterization studies revealed that these type 3 derivatives were indistinguishable from the type 2, type 5, and type 6B parental strains in terms of restriction enzyme fragment pattern and expression of pneumococcal surface protein A (PspA). Expression of the type 3 capsule did not alter the mouse virulence of the similarly virulent type 2 strain. However, alteration of capsule type had a profound effect on the virulence of the type 5 and type 6B derivatives. The highly virulent type 5 strain was essentially avirulent when expressing the type 3 capsule. Conversely, the 50% lethal dose of the relatively avirulent type 6B strain was reduced > 100-fold when the type 3 capsule was expressed. Thus, the serotype of capsule expressed has a major effect on virulence, and this effect is dependent upon the genetic background of the recipient strain.

Outbreak of methicillin-resistant Staphylococcus aureus in a Teaching Hospital — epidemiological and microbiological surveillance

An outbreak of methicillin-resistant S. aureus (MRSA) in a large university teaching hospital occurred between December 1991 and May 1992, involving 7 different wards and more than 30 patients. Epidemiological typing was performed to control the epidemic and to identify the MRSA carriers. By a combination of various classical methods (antimicrobial susceptibility, phage typing) and molecular typing procedures (SDS-PAGE of extracellular proteins, plasmid DNA profile, restriction enzyme fragment pattern of chromosomal DNA), three different clones of MRSA could be discriminated. The epidemic clone A was recovered from 30 patients and from 3 staff members. By strict microbiological monitoring together with hygienic measures, the epidemic could be successfully controlled. It is concluded that a combination of phenotypic markers and DNA-based epidemiological markers is extremely useful in the microbiological surveillance of MRSA outbreaks.

DNA restriction endonuclease profiles and typing of geographically diverse isolates ofBacillus larvae

SUMMARYDNA isolated from each of 20 Bacillus larvae isolates of geographically diverse origin (17 from Australia, one each from Fiji, Mexico and New Zealand) and cultured on brain heart infusion agar was digested with a range of restriction endonucleases. Based upon the degree of similarity in restriction endonuclease fragment patterns (REFP) produced by the restriction endonuclease Cfo I, five clonal types of B. larvae were identified. REFP were resolved on 3.5% Polyacrylamide gels and visualized by silver staining. All the isolates showed many similarities in their REFP, however, those isolates cultured from geographically localized regions showed the greatest similarity. The superior resolving capabilities of polyacrylamide enabled these isolates to be further sub-typed according to minor variations in REFP.