Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

Background  

Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes.     

Campylobacter fetus subspecies venerealis surface array protein from bovine isolates in Brazil

The electrophoretic patterns of 31 Campylobacter fetus subspecies venerealis capsular Surface Array Protein (SAP) isolated from bovines in reproduction from different regions of Brazil were analyzed. The persistence of the bacteria in the reproductive tract of naturally infected bovines and the dynamic of SAP expression were also evaluated. Cervical mucous and prepucial aspirates from five animals naturally infected were cultured for isolation of Campylobacter fetus and the SAPs extracted from the bacteria isolated were analyzed by SDS-PAGE. Ten different patterns of SAP expression were demonstrated by the identification of proteins with molecular mass of 97, 100, 127, and 149 kDa, respectively. The most prevalent identified protein had a molecular mass of 100 kDa (41.9%). Taking into consideration the time during which the five animals were evaluated, it was possible to conclude that one of these animals persisted with the etiological agent up to 171 days. The five naturally infected bovines analyzed presented variation on their surface protein pattern during the period of this study. C. fetus subspecies venerealis persisted in the reproductive tract of naturally infected animals. In natural condition of infection C. fetus subspecies venerealis persisted in an intermittent condition and an alteration of the protein surface was shown. Received: 27 August 2001 / Accepted: 4 December 2001     

Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non‐motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup‐like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup‐like structure composed of two membranes.     

Real Time PCR to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis

Bovine venereal campylobacter infection, caused by Campylobacter fetus venerealis, is of significant economic importance to the livestock industry. Unfortunately, the successful detection and discrimination of C. fetus venerealis from C. fetus fetus continue to be a limitation throughout the world. There are several publications warning of the problem with biotyping methods as well as with recent molecular based assays. In this study, assessed on 1071 isolates, we report on the successful development of two Real Time SYBR® Green PCR assays that will allow for the detection and discrimination of C. fetus fetus and C. fetus venerealis. The sensitivity reported here for the C. fetus (CampF4/R4) and the C. fetus venerealis (CampF7/R7) specific PCR assays are 100% and 98.7% respectively. The specificity for these same PCR assays are 99.6% and 99.8% respectively.     

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking.     

Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1β, TNF-α, and IL-8. In this study, the expression of IL-1β, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1β and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1β and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.     

Identification of catalase-producingCampylobacter species based on biochemical characteristics and on cellular fatty acid composition

A total of 50 catalase-positive campylobacters from human and animal sources were studied. The nomenclatural type strains ofCampylobacter coli, C. jejuni, C. fetus, andC. fetus subsp.venerealis, a typical strain of the nalidixic acid-resistant thermophilic group, and various clinical isolates were characterized by bacteriological tests and by gas-liquid chromatographic analysis of their cellular fatty acids. The tests most useful in the differentiation of the various catalase-positive species were growth at 25 and 42°C, H₂S production, tolerance to nalidixic acid and to 2,3,5-triphenyltetrazolium chloride, and hippurate hydrolysis. The latter test was the only reliable means to differentiate betweenC. coli andC. jejuni. Differences between.C. fetus andC. jejuni/coli were confirmed by cellular fatty acid compositions. The bacteriological results indicated thatC. fetus andC. jejuni were distinct species, although within the thermophilic campylobacters there were several related taxa that included bothC. coli andC. jejuni strains with typicalC. coli and some thermophilic strains ofC. fetus subsp.fetus at the extremes.     

Development of Experimental Genetic Tools for Campylobacter fetus

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.     

Morphological Differences in Flagella in Campylobacter fetus Subsp. intestinalis and C. fetus Subsp. jejuni

Intact flagella were isolated from human pathogenic strains of Campylobacter, C. fetus subsp. intestinalis and C. fetus subsp. jejuni, by the method of DePamphilis and Adler and examined by electron microscopy. The isolated flagella were composed of a filament, a hook, a basal body, and a large disk associated with the end of the hook region covering the basal body. The width of the hook was approximately 28 nm, somewhat greater than that of the filament (20 nm in diameter). The hook region of C. fetus subsp. intestinalis was curved, but it was straight in C. fetus subsp. jejuni. The structure of the basal body of the two subspecies was similar to that reported for other gram-negative bacteria. The large disk detached from the flagella showed concentrically arranged circular structures. This structure was more clearly observed in the disk of C. fetus subsp. jejuni than in C. fetus subsp. intestinalis. Observations of thin-sectioned profiles at the attachment site of the flagellum revealed that the large disk is located on the inner side of the outer membrane. The role of the large disk in bacterial movement is not clear, but it is assumed that it acts as an organ to protect the flagellar insertion site from vigorous rotation of the polar end inflicted during bacterial movement.     

Molecular mechanisms of Campylobacter fetus surface layer protein expression

Cells of the Gram-negative bacteria Campylobacter fetus are covered by monomolecular arrays of surface layer proteins (SLPs) critical for both persistence in their natural hosts and for virulence. For C. fetus cells, expression of SLPs essentially eliminates C3b binding and their antigenic variation thwarts host immunological defences. Each cell possesses multiple partially homologous and highly conserved SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97–149 kDa. These attach non-covalently via a conserved N-terminus to the cell wall lipopolysaccharide. Recent studies indicate that C. fetus reassorts a single promoter, controlling SLP expression, and one, or more, complete open reading frame strictly by DNA inversion, and that rearrangement is independent of the distance between sites of inversion. In contrast to previously reported programmed DNA inversion systems, inversion in C. fetus is recA- dependent. These rearrangements permit variation in protein expression from the family of SLP genes and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.     

Comparative Genome Analysis of Campylobacter fetus Subspecies Revealed Horizontally Acquired Genetic Elements Important for Virulence and Niche Specificity

Campylobacter fetus are important animal and human pathogens and the two major subspecies differ strikingly in pathogenicity. C. fetus subsp. venerealis is highly niche-adapted, mainly infecting the genital tract of cattle. C. fetus subsp. fetus has a wider host-range, colonizing the genital- and intestinal-tract of animals and humans. We report the complete genomic sequence of C. fetus subsp. venerealis 84-112 and comparisons to the genome of C. fetus subsp. fetus 82-40. Functional analysis of genes predicted to be involved in C. fetus virulence was performed. The two subspecies are highly syntenic with 92% sequence identity but C. fetus subsp. venerealis has a larger genome and an extra-chromosomal element. Aside from apparent gene transfer agents and hypothetical proteins, the unique genes in both subspecies comprise two known functional groups: lipopolysaccharide production, and type IV secretion machineries. Analyses of lipopolysaccharide-biosynthesis genes in C. fetus isolates showed linkage to particular pathotypes, and mutational inactivation demonstrated their roles in regulating virulence and host range. The comparative analysis presented here broadens knowledge of the genomic basis of C. fetus pathogenesis and host specificity. It further highlights the importance of surface-exposed structures to C. fetus pathogenicity and demonstrates how evolutionary forces optimize the fitness and host-adaptation of these pathogens.     

A Genomic Island Defines Subspecies-Specific Virulence Features of the Host-Adapted Pathogen Campylobacter fetus subsp. venerealis

The pathogen Campylobacter fetus comprises two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis. Although these taxa are highly related on the genome level, they are adapted to distinct hosts and tissues. C. fetus subsp. fetus infects a diversity of hosts, including humans, and colonizes the gastrointestinal tract. In contrast, C. fetus subsp. venerealis is largely restricted to the bovine genital tract, causing epidemic abortion in these animals. In light of their close genetic relatedness, the specific niche preferences make the C. fetus subspecies an ideal model system to investigate the molecular basis of host adaptation. In this study, a subtractive-hybridization approach was applied to the genomes of the subspecies to identify different genes potentially underlying this specificity. The comparison revealed a genomic island uniquely present in C. fetus subsp. venerealis that harbors several genes indicative of horizontal transfer and that encodes the core components necessary for bacterial type IV secretion. Macromolecular transporters of this type deliver effector molecules to host cells, thereby contributing to virulence in various pathogens. Mutational inactivation of the putative secretion system confirmed its involvement in the pathogenicity of C. fetus subsp. venerealis.Campylobacter species are Gram-negative epsilonproteobacteria highly adapted to mucosal surfaces. The majority are human and/or animal pathogens (19, 61). The 18 species comprising the genus Campylobacter display a high degree of host and tissue specificity, which makes them excellent models to study host-pathogen relationships (25). The most prominent member, Campylobacter jejuni, is a commensal of the chicken intestine and the major cause of human bacterial diarrhea (74). Comparative analysis of Campylobacter genomes has revealed a process of genome decay—supported by a small genome size (about 1.5 Mb) and the loss of metabolic genes—consistent with successful adaptation to a specific niche (41). Campylobacter genomes are among the densest bacterial genomes known, with about 95% coding sequence. Despite this evidence of reduction, plasticity in genetic composition remains evident, as strain-specific genes comprise a substantial proportion of the entire repertoire of 1,500 to 1,800 genes (16, 23, 25, 56).This study focuses on the species Campylobacter fetus, which is represented by the two subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. Although the two taxa are genetically closely related, they exhibit striking tissue and host specificity. C. fetus subsp. fetus is a human, as well as animal, pathogen. Human infection results in serious systemic disease, especially in immunocompromised people. C. fetus subsp. fetus is the Campylobacter species most often isolated from human blood (75), and it is considered an emerging pathogen (9). The infection mode shares similarities with that of Salmonella enterica serovar Typhi. Orally acquired C. fetus subsp. fetus penetrates the intestinal mucosa, leading to bacteremia, and subsequent excretion via the biliary tract leads to secondary colonization of the intestine (9). Colonization of reproductive organs induces abortion in sheep and to a lesser extent in cattle, and very rarely in humans (11). C. fetus subsp. fetus can also be isolated from the intestinal tracts of birds and reptiles (78, 80). In contrast, C. fetus subsp. venerealis is host restricted. It is isolated primarily from the bovine genital tract and causes the epidemic disease bovine venereal campylobacteriosis (BVC). The reservoir of C. fetus subsp. venerealis is the penile prepuce of the bull. Transmission to cows occurs at coitus or during artificial insemination, and infection leads to endometritis, abortion, and infertility (28). Since BVC is a worldwide problem with substantial economic consequences, diagnosed cases must be registered (75) and import and export of bovine semen and embryos for cattle breeding requires statutory preclusion of C. fetus infection (2). Despite the distinct niche preferences of the C. fetus subspecies, they show high genetic relatedness, complicating the task of correct subspecies identification (46, 62, 81). Their population structure is clonal, and C. fetus subsp. venerealis is thought to represent a bovine clone of C. fetus (81).In this study, we employed the C. fetus subspecies to investigate the genetic basis for their host and tissue specificities. A genomic subtractive-hybridization approach was taken to identify subspecies-specific genomic fragments. This led to the discovery of a genomic island exclusively present on the chromosome of the host-adapted subspecies C. fetus subsp. venerealis. This island harbors a type IV secretion system (T4SS), as well as mobility genes (insertion sequence [IS] transposases and phage integrases) and shares substantial homology and similar structure with resistance plasmids found in other Campylobacter species. These features are indicative of a horizontally acquired genetic element. Finally, mutational analysis of genes within the island substantiates its involvement in C. fetus subsp. venerealis virulence.     

Southern blotting analyses of strains ofCampylobacter fetus using the conserved region ofsapA

Chromosomal DNA of 27 strains of Campylobacter fetus was analyzed by Southern blotting with a probe of the conserved region of sapA. The probe hybridized with 23 strains that produced type A lipopolysaccharide. These strains had more than six sapA homologs. In Southern blots of SalI-digested chromosomal DNA separated by pulsed-field gel electrophoresis, one fragment from 19 strains and two fragments from 4 strains hybridized. These data indicate that multiple sapA homologs are localized to a limited region on the chromosomal DNA of C. fetus and thus suggest the possibility of developing a typing system using this method. Received: 28 June 1995 / Accepted: 19 September 1995     

Generation of Campylobacter fetus S-layer protein diversity utilizes a single promoter on an invertible DNA segment

Wild-type strains of Campylobacter fetus contain a monomolecular array of surface layer proteins (SLPs) and vary the antigenicity of the predominant SLP expressed. Reciprocal recombination events among the eight genomic SLP gene cassettes, which encode 97- to 149 kDa SLPs, permit this variation. To explore whether SLP expression utilizes a single promoter, we created mutant bacterial strains using insertional mutagenesis by rescue of a marker from plasmids. Experimental analysis of the mutants created clearly indicates that SLP expression solely utilizes the single sapA promoter, and that for variation C. fetus uses a mechanism of DNA rearrangement involving inversion of a 6.2 kb segment of DNA containing this promoter. This DNA inversion positions the sapA promoter immediately upstream of one of two oppositely oriented SLP gene cassettes, leading to its expression. Additionally, a second mechanism of DNA rearrangement occurs to replace at least one of the two SLP gene cassettes bracketing the invertible element. As previously reported promoter inversions in prokaryotes, yeasts and viruses involve alternate expression of at most two structural genes, the ability of C. fetus to use this phenomenon to express one of multiple cassettes is novel.     

High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis

Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full-length S-layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10^-2. Immunoblots of survivors showed that phenotypic reversion involving high-level production of full-length (98, 127 or 149 kDa) S-layer proteins had occurred. Revertants were serum resistant but caused approximately 10-fold less bacteraemia in orally challenged mice than did the wild-type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high-frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.     

The occurrence of Campylobacter in river water and waterfowl within a watershed in southern Ontario,Canada

Aims: Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. Methods and Results: Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (<130 cells 100 ml^?1) in 57–79% of samples taken from five locations. By comparison, a culture‐based method detected Campylobacter in 0–23% of samples. Water quality parameters such as total Escherichia coli were not highly correlated with Campylobacter levels, although higher pathogen concentrations were observed at colder water temperatures (<10°C). Strains isolated from river water were primarily nalidixic acid‐susceptible Campylobacter lari, and selected isolates were identified as Campylobacter lari ssp. concheus. Campylobacter from wild birds (seagulls, ducks and geese) were detected at a similar rate using PCR (32%) and culture‐based (29%) methods, and although Campylobacter jejuni was isolated most frequently, C. lari ssp. concheus was also detected. Conclusions: Campylobacter were frequently detected at low concentrations in the watershed. Higher prevalence rates using quantitative PCR was likely because of the formation of viable but nonculturable cells and low recovery of the culture method. In addition to animal and human waste, waterfowl can be an important contributor of Campylobacter in the environment. Significance and Impact of the Study: Results of this study show that Campylobacter in surface water can be an important vector for human disease transmission and that method selection is important in determining pathogen occurrence in a water environment.     

Mismatch of morphological and molecular identifications in native and invasive subspecies of Codium fragile (Bryopsidophyceae,Chlorophyta)

Several subspecies are defined within Codium fragile, including the invasive C. fragile ssp. fragile, first reported in New Zealand in 1973. An endemic subspecies, C. fragile ssp. novae‐zelandiae, is also found throughout New Zealand. The two subspecies exhibit morphological and molecular variation, although these have never been evaluated together. We compared variation between subspecies at locations in Auckland, identifying subspecies using rps3‐rpl16 DNA sequence data, and assessing gross morphological differences, anatomical utricle characters and morphometrics. The taxonomic utility of the morphometric data sets was assessed by linear discriminant analysis. Utricle characters and measurements varied within individual thalli and between different preservation methods. The phenotypes of both subspecies were highly variable and influenced by environment. Accurate subspecies delimitation using morphological data was not possible; the discriminant analyses performed no better than chance for all combinations of the morphological data. Specimens from New Zealand, Canada, Australia and Ireland were sequenced using both the rps3‐rpl16 and tufA plastid markers. The tufA elongation factor was shown to be a good candidate for differentiating subspecies of C. fragile. This marker is twice the length of the rps3‐rpl16 spacer, shows greater variation between ssp. fragile and novae‐zelandiae, and is less prone to sequencing error. A simple restriction enzyme digest of the tufA amplicon can distinguish ssp. fragile and ssp. novae‐zelandiae. Our study expands the known range of the ssp. fragile in New Zealand, including the first record of this subspecies from the west coast of Auckland, and points to a need to re‐evaluate morphological and molecular criteria for subspecies currently defined within C. fragile.     

Development of loop‐mediated isothermal amplification and PCR assays for rapid and simple detection of Campylobacter fetus subsp. venerealis

Campylobacter fetus is divided into CFV and CFF. Because CFV causes bovine genital campylobacteriosis, differentiation of the two subspecies is essential to the implementation of efficient CFV control and eradication programs. We have developed LAMP and duplex PCR assays for rapid and simple detection of CFV. The LAMP assay correctly detected 7 CFV strains and did not detect 53 CFF, 35 non‐fetus Campylobacter and 25 non‐Campylobacter strains. The PCR assay successfully differentiated the two subspecies. The LAMP and PCR assays were faster than conventional biochemical assays, requiring for detection less than 50 min and less than 4 hr, respectively, from the beginning of DNA extraction from a single colony on blood agar to final determination. Our LAMP and PCR assays are rapid and practical tools for detection of CFV.     

An environmentally regulated pilus-like appendage involved in Campylobacter pathogenesis

Examination of strains of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by electron microscopy revealed that they produced peritrichous pilus-like appendages when the bacteria were grown in the presence of bile salts. Various bile-salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 μm in length. A gene, termed pspA, which encodes a predicted protein resembling protease IV of Escherichia coli, was identified in C. jejuni strain 81–176. A site-specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non-piliated mutant of strain 81–176 showed no reduction in adherence to or invasion of INT 407 cells in vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.     

Campylobacter fetus septic arthritis: report of a case.

We report a case of septic arthritis caused by the fastidious gram-negative rod Campylobacter fetus. We suggest that the organism may be part of the endogenous flora and that the clinical infections tend to occur in compromised hosts. Our patient is the first to be described with multiple myeloma and C. fetus septic arthritis. The documented cases of culture-proven C. fetus septic arthritis reported to date have occurred in three men and one woman, all in the seventh and eighth decades of life, with a mono-articular large joint distribution. The septic arthritis always occurred in previously injured joints and curiously enough need not be associated with a toxic-appearing patient. C. fetus infections are also associated with the signs and symptoms of clinical thrombophlebitis. We stress caution in establishing this diagnosis of phlebitis on clinical evaluation only and urge differentiation of true deep vein thrombophlebitis from pseudothrombophlebitis or dissected popliteal synovial cyst. This latter diagnosis may be made non-invasively by ultrasound techniques.